Fuyuki Sato

DEC1 negatively regulates AMPK activity via LKB1

Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) is one of the clock genes that show a circadian rhythm in various tissues. AMP-activated protein kinase (AMPK) activity plays important roles in the metabolic process and in cell death induced by glucose depletion. Recent reports have shown that AMPK activity exhibited a circadian rhythm. However, little is known regarding the regulatory mechanisms involved in the circadian rhythm of AMPK activity. The aim of this study is to investigate whether there is a direct correlation between DEC1 expression and AMPK activity. DEC1 protein and AMPK activity showed a circadian rhythm in the mouse liver with different peak levels. Knocking down DEC1 expression increased AMPK activity, whereas overexpression of DEC1 decreased it. Overexpressing the DEC1 basic mutants had little effect on the AMPK activity. DEC1 bound to the E-box of the LKB1 promoter, decreased LKB1 activity and total protein levels. There was an inverse relationship between DEC1 expression and AMPK activity. Our results suggest that DEC1 negatively regulates AMPK activity via LKB1.

Smad3 plays an inhibitory role in phosphate-induced vascular smooth muscle cell calcification

Arterial medial calcification is a major complication in patients with chronic kidney disease and diabetes. It has been hypothesized that a high concentration of inorganic phosphate (Pi) induces calcification in vascular smooth muscle cells (vSMCs). However, the role of transforming growth factor-β (TGF-β)/Smad3 signaling in Pi-induced vascular calcification remains controversial. The aim of this study was to investigate the possible involvement of Smad3 in Pi-induced vascular calcification. We compared the degree of Pi-induced vSMC calcification between vSMCs isolated from wild-type (Smad3(+/+)) and Smad3-deficient (Smad3(-/-)) mice. We found that vSMCs from Smad3(+/+) mice had less calcium (Ca) than those from Smad3(-/-) mice when they were exposed to high concentrations of Pi and Ca (Pi+Ca). The phosphorylation of Smad3 was induced in Smad3(+/+) vSMCs by exposure to Pi+Ca. The concentration of extracellular pyrophosphate (ePPi) was lower in Smad3(-/-) vSMCs than in Smad3(+/+) vSMCs and was significantly increased in Smad3(+/+) vSMCs by treatment with TGF-β1. Also, the addition of a small amount of PPi to culture medium significantly decreased the deposition of Ca in both Smad3(+/+) and Smad3(-/-) vSMCs. Ectonucleotide phosphatase/phosphodiesterase1 (Enpp1) was decreased at the mRNA, protein, and enzymatic activity levels in Smad3(-/-) vSMCs compared with Smad3(+/+) vSMCs. A ChIP assay showed that phosphorylated Smad3 directly binds to the Enpp1 gene. Furthermore, the calcification of aortic segments was attenuated by treatment with TGF-β1 only in Smad3(+/+) mice. Taken together, we conclude that Pi-induced vSMC calcification is suppressed by Smad3 via an increase in ePPi.

The basic helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates Twist1 through an E-box element.

Differentiated embryo chondrocyte 2 (DEC2/Sharp-1/Bhlhe41), a basic helix-loop-helix (bHLH) transcription factor, has been shown to regulate the transcription of target genes by binding to their E-box elements. We identified a possible DEC2-response element (consensus E-box: CACGTG) in the promoter region of Twist1. Forced expression of DEC2 significantly repressed Twist1 promoter activity under normoxia and under hypoxia as assessed by a luciferase reporter assay. In addition, over-expression of DEC2 repressed Twist1 mRNA expression assessed by quantitative real-time PCR. Site-directed mutagenesis studies showed that mutagenesis of the consensus E-box sequence eliminated the ability of DEC2 to reduce the Twist1 promoter activity. Chromatin immunoprecipitation (ChIP) assays confirmed that the DEC2-mediated repression is primarily achieved by binding to the E-box in the Twist1 promoter. Knockdown of DEC2 by siRNA significantly attenuated the repression of Twist1 expression. DEC2 and Twist1 exhibit inversed protein expression patterns during development of mouse tongue embryo tissue. Given the fact that DEC2 protein is emerging as an important regulator in a vast array of cellular events, including cell differentiation, maturation of lymphocytes and the molecular clock, our study elucidates an important mechanism by which DEC2 regulates cellular function by modulating the expression of Twist1.

Impact of heart-specific disruption of the circadian clock on systemic glucose metabolism in mice

ABSTRACT The daily rhythm of glucose metabolism is governed by the circadian clock, which consists of cell-autonomous clock machineries residing in nearly every tissue in the body. Disruption of these clock machineries either environmentally or genetically induces the dysregulation of glucose metabolism. Although the roles of clock machineries in the regulation of glucose metabolism have been uncovered in major metabolic tissues, such as the pancreas, liver, and skeletal muscle, it remains unknown whether clock function in non-major metabolic tissues also affects systemic glucose metabolism. Here, we tested the hypothesis that disruption of the clock machinery in the heart might also affect systemic glucose metabolism, because heart function is known to be associated with glucose tolerance. We examined glucose and insulin tolerance as well as heart phenotypes in mice with heart-specific deletion of Bmal1, a core clock gene. Bmal1 deletion in the heart not only decreased heart function but also led to systemic insulin resistance. Moreover, hyperglycemia was induced with age. Furthermore, heart-specific Bmal1-deficient mice exhibited decreased insulin-induced phosphorylation of Akt in the liver, thus indicating that Bmal1 deletion in the heart causes hepatic insulin resistance. Our findings revealed an unexpected effect of the function of clock machinery in a non-major metabolic tissue, the heart, on systemic glucose metabolism in mammals.

Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts via TNF-α

Bmal1, a clock gene, is associated with depression, hypertrophy, metabolic syndrome and diabetes. Smad3, which is involved in the TGF-β signaling pathway, plays an important role in the regulation of tumor progression, fibrosis, obesity and diabetes. Our previous report showed that Smad3 has circadian expression in mouse livers. In the current study, we focused on the heart, especially on the myocardial stromal fibroblasts because the roles of Bmal1 and Smad3 in this tissue are poorly understood. Bmal1 and Smad3 have circadian expression in mouse hearts, and their circadian expression patterns were similar. Bmal1 expression decreased in the hearts of whole-body Smad3 knockout mice, whereas Smad3 expression had little effect on heart-specific Bmal1 knockout mice. Both Smad3 knockout and heart-specific Bmal1 knockout mice showed increases in p21, S100A4, CD206 and TNF-α expression in the myocardial stromal fibroblasts and macrophage compared to control mice. We also examined Smad3, Bmal1 and Dec1 expression in human tissue from old myocardial infarctions. Expression of Smad3, Bmal1 and Dec1 decreased in the stromal fibroblasts of tissue from old myocardial infarctions compared to control cases. On the other hand, p21, S100A4 and TNF-α increased in the stromal fibroblasts of tissue from old myocardial infarctions. Furthermore, expression of Smad3, Bmal1 and Dec1 decreased in TNF-α treated-NIH3T3 cells but expression of p21 and S100A4 increased. This new evidence suggests that Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts through TNF-α.

Abstract 2026: DEC1 negatively regulates AMPK activity via LKB1

Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) is one of the clock genes that show a circadian rhythm in various tissues. AMP-activated protein kinase (AMPK) activity plays important roles in metabolism and in cell death induced by glucose depletion. Recent reports have shown that AMPK activity exhibited a circadian rhythm. However, how the circadian rhythm of AMPK activity is regulated is not known. The aim of this study is the direct correlation between DEC1 expression and AMPK activity. DEC1 protein and AMPK activity showed a circadian rhythm in the mouse liver with different peak levels. A medium change and serum shock led to rhythmic patterns of DEC1 protein and AMPK activity levels in WI-38 cells, and their peak levels occurred at different times. However, changing the medium did not induce circadian rhythms of DEC1 protein or AMPK activity levels in MCF-7 and U2OS cells, in which the intensities of these levels had inversely correlated patterns. Knocking down DEC1 expression increased AMPK activity, whereas DEC1 overexpression decreased it. DEC1 bound to the E-box of the LKB1 promoter, decreasing the LKB1 activity and total protein levels. In addition, knocking down DEC1 expression inhibited cell death under the condition of glucose depletion, increasing the activity of both AMPK and LKB1. Taken together, our results showed that the levels of DEC1 protein and AMPK activity were affected even long after the medium change and that the rhythmic patterns of these levels were inversely correlated. We concluded that DEC1 negatively regulated AMPK activity via LKB1. Citation Format: Fuyuki Sato, Yasuteru Muragaki. DEC1 negatively regulates AMPK activity via LKB1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2026.

Abstract 2999: DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression

Circadian rhythms are tightly regulated by clock genes. It has been reported that clock genes plays important roles in biological function of normal cells. However, it is not well understood the function of clock genes in tumor cells. We showed that basic helix-loop-helix (bHLH) transcription factors, differentiated embryonic chondrocyte gene 1 (DEC1/BHLHE40/Sharp2/Stra13) and DEC2 (BHLHE41/Sharp1), are clock genes and play important roles in circadian rhythm, cell proliferation, apoptosis, hypoxia response, various stresses and epithelial-to-mesenchymal transition (EMT) in tumor cells. Various stresses, such as exposure to transforming growth factor-beta (TGF-β), hypoxia, cytokines, serum-free, and anti-tumor drugs affect DEC expression. The increased or decreased DEC expression by such stresses is involved in tumor progression. The evidence that DEC2 has circadian expression in implanted mouse sarcoma cells and negatively regulates VEGF expression under hypoxia. DEC1 has pro-apoptotic effects in human breast cancer cells, whereas DEC2 has anti-apoptotic effects. DEC1 induced EMT by TGF-β in human pancreatic cancer cells, whereas DEC2 suppressed EMT. Immunohistochemical analysis showed that DEC1 is highly expressed in tumor cells and the expression is involved in malignancy. On the other hand, DEC2 is little expressed in tumor cells compared with adjacent non-tumor cells. These findings suggest that DEC1 and DEC2 have different roles in tumor progression under circadian rhythm. DEC2 regulates the circadian rhythm and VEGF expression by negative feed-back system, and DEC1 and DEC2 regulates EMT by positive and negative feed-back system in tumor cells. So, DEC acts as “accelerator” and “brake” in tumor progression. In this study, we show current progress of the DEC genes in tumor progression. Citation Format: Fuyuki Sato, Yasuteru Muragaki. DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2999. doi:10.1158/1538-7445.AM2015-2999

Trps1 deficiency inhibits the morphogenesis of secondary hair follicles via decreased Noggin expression

A representative phenotype of patients with tricho-rhino-phalangeal syndrome (TRPS) is sparse hair. To understand the developmental defects of these patients hair follicles, we analyzed the development of hair follicles histologically and biochemically using Trps1 deficient (KO) mice. First, we compared the numbers of primary hair follicles in wild-type (WT) and KO embryos at different developmental stages. No differences were observed in the E14.5 skins of WT and KO mice. However, at later time points, KO fetal skin failed to properly develop secondary hair follicles, and the number of secondary hair follicles present in E18.5 KO skin was approximately half compared to that of WT skin. Sonic hedgehog expression was significantly decreased in E17.5 KO skin, whereas no changes were observed in Eda/Edar expression in E14.5 or E17.5 skins. In addition, Noggin expression was significantly decreased in E14.5 and E17.5 KO skin compared to WT skin. In parallel with the suppression of Noggin expression, BMP signaling was promoted in the epidermal cells of KO skins compared to WT skins as determined by immunohistochemistry for phosphorylated Smad1/5/8. The reduced number of secondary hair follicles was restored in skin graft cultures treated with a Noggin and BMP inhibitor. Furthermore, decreased cell proliferation, and increased apoptosis in KO skin was rescued by Noggin treatment. Taken together, we conclude that hair follicle development in Trps1 KO embryos is impaired directly or indirectly by decreased Noggin expression.