Circadian regulation of low density lipoprotein receptor promoter activity by CLOCK/BMAL1, Hes1 and Hes6

Abstract

Low density lipoprotein receptor (LDLR) plays an important role in the cholesterol homeostasis. We examined the possible circadian regulation of LDLR and mechanism(s) underlying it. In mice, blood glucose and plasma triglyceride, total and high density lipoprotein cholesterol varied distinctively throughout a day. In addition, LDLR mRNA oscillated in the liver in a functional clock-dependent manner. Accordingly, analysis of human LDLR promoter sequence revealed three putative E-boxes, raising the possible regulation of LDLR expression by E-box-binding transcription factors. To test this possibility, human LDLR promoter reporter constructs were transfected into HepG2 cells and the effects of CLOCK/BMAL1, Hes1, and Hes6 expression were analyzed. It was found that positive circadian transcription factor complex CLOCK/BMAL1 upregulated human LDLR promoter activity in a serum-independent manner, while Hes family members Hes1 and Hes6 downregulated it only under serum-depleted conditions. Both effects were mapped to proximal promoter region of human LDLR, where mutation or deletion of well-known sterol regulatory element (SRE) abolished only the repressive effect of Hes1. Interestingly, hes6 and hes1 mRNA oscillated in an anti-phasic manner in the wild-type but not in the per1-/-per2 -/- mouse. Comparative analysis of mouse, rat and human hes6 genes revealed that three E-boxes are conserved among three species. Transfection and site-directed mutagenesis studies with hes6 reporter constructs confirmed that the third E-box in the exon IV is functionally induced by CLOCK/BMAL1. Taken together, these results suggest that LDLR expression is under circadian control involving CLOCK/BMAL1 and Hes family members Hes1 and Hes6.

Figure 1

Daily rhythms of metabolic parameters related to cholesterol homeostasis in mice fed ad libitum. (A-D) Male C57BL/6 mice were entrained to 12L:12D cycle for 2 weeks and sacrificed by cervical dislocation at the indicated zeitgeber time (ZT) and whole blood samples were collected. Blood glucose (A), plasma TG (B), total cholesterol (C) and HDL cholesterol were determined by specific kits obtained from Callegrari™. Data are expressed as mean ± S.E.M. (n = 8). (E) Oscillation of ldlr mRNA in the mouse liver. Male C57BL/6 mice were entrained to 12L:12D cycle for 2 weeks and released to DD. On the second day after light-off, mice were sacrificed by cervical dislocation at the indicated circadian time and liver samples were obtained. RNA isolation, reverse transcription, and real-time polymerase chain reaction were performed to measure specific messages for mouse ldlr. All mRNA levels were normalized to gapdh mRNA level and expressed as mean ± S.E.M. (n = 8).

Figure 2

Transcriptional regulation of ldlr promoter activity by CLOCK/BMAL1, Hes1 and Hes6. (A) Schematic diagram of DNA elements on human ldlr gene promoter and serial deletion constructs. Genomic DNA sequences spanning human ldlr (NT_011295, chromosome 19) were analyzed for the transcription factor binding sites using tools provided by Genomatrix (www.genomatrix.de). DNA elements were symbolized as indicated in the figure. The predicted transcription start site is indicated as a bent arrow. Schematic drawing of human ldlr gene reporter constructs is also shown in the lower part of the figure. (B, C) Effect of CLOCK/BMAL1, Hes1 and Hes6 expression on reporter activity. HepG2 human hepatocarcinoma cells were transiently transfected with each reporter construct with or without CLOCK/BMAL1, Hes1, or Hes6 expression vectors. One day after transfection, cells received either 10% FBS-containing (B) or serum-depleted media (C). Luciferase assay was performed after additional 24 h incubation. Resulting firefly luciferase activity was normalized to Renilla luciferase activity driven by TK promoter, which was used as an internal control. Data are expressed as mean ± S.E.M. (n = 3). (D) The sterol regulatory element (SRE) in the proximal promoter of human ldlr gene is responsible for inhibition by HES1. Effect of CLOCK/BMAL1, Hes1 and Hes6 on reporter activity. HepG2 cells were transiently transfected with pLR 1563-luc or pLR 1563 mSRE-luc or pLR 1563 dSRE-luc construct with or without CLOCK/BMAL1, Hes1 and Hes6 expression vectors. Resulting firefly luciferase activity was normalized to Renilla luciferase activity driven by TK promoter, which were used as an internal control. Data are expressed as mean ± S.E.M.

Figure 3

Anti-phasic oscillation of hes1 and hes6 mRNA in the wild-type (A) but not in per1^-/-per2^-/- (B) mouse liver. Male C57BL/6 mice of each genotype were entrained to 12L:12D cycle for 2 weeks and released to DD. On the second day after light-off, mice were sacrificed by cervical dislocation at the indicated circadian time and liver samples were obtained. RNA isolation, reverse transcription, and real-time polymerase chain reaction were performed to measure specific messages for rev-erba, cry1, bmal1b, hes6 and hes1. All mRNA levels were normalized to gapdh mRNA level and expressed as mean ± S.E.M. (n = 3).

Figure 4

Mouse hes6 gene is induced by CLOCK/BMAL1 via downstream regulatory sequence. (A) Schematic diagrams of mouse, rat, and human hes6 gene structure and conserved DNA elements. Genomic DNA sequences spanning mouse (Accession No. NT078297, chromosome 1), rat (NW_047817, chromosome 9), and human hes6 (NT_005120, chromosome 2) were analyzed for the common transcription factor binding sites using tools provided by Genomatrix (www.genomatrix.de). Evolutionarily conserved DNA elements were symbolized as indicated in the figure. (B) Schematic drawing of mouse hes6 gene reporter constructs. The four serial deletion constructs were made from upstream promoter region of hes6 and regulatory elements were made from intergenic region. See Materials and Methods for details. The right headed triangle indicates SV40 promoter. (C, D) Effect of CLOCK/BMAL1 expression on reporter activity. NIH 3T3 mouse fibroblast (C) and HT22 mouse hippocampal cells (D) were transiently transfected with each reporter construct with or without CLOCK/BMAL1 expression vectors. Resulting firefly luciferase activity was normalized to Renilla luciferase activity driven by TK promoter, which was used as an internal control. Data are expressed as mean ± S.E.M.

Figure 5

Conserved E-box in the exon IV of mouse hes6 gene is responsible for induction by CLOCK/BMAL1 and inhibition by CRY1. (A) Location of conserved E-box in HireB-Luc construct and point mutation resulting in HireBmut-Luc construct. Large triangle indicates SV40 promoter. (B, C) Effect of CLOCK/BMAL1 and increasing amount of CRY1 expression on reporter activity. NIH 3T3 mouse fibroblasts (B) and HT22 mouse hippocampal cells (C) were transiently transfected with either HireB-Luc or HireBmutE-Luc construct with or without CLOCK/BMAL1 and CRY1 expression vectors. Resulting firefly luciferase activity were normalized to Renilla luciferase activity driven by TK promoter, which were used as an internal control. Data are expressed as mean ± S.E.M.