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A molecular clock regulates angiopoietin-like protein 2 expression.

Kadomatsu T, Uragami S, Akashi M, Tsuchiya Y, Nakajima H, Nakashima Y, Endo M, Miyata K, Terada K, Todo T, Node K, Oike Y - PLoS ONE (2013)

Bottom Line: Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression.We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells.Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. tkado@gpo.kumamoto-u.ac.jp

ABSTRACT
Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

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Non-canonical E-boxes mediate CLOCK/BMAL1-dependent induction of human ANGPTL2 promoter activity.A, Schematic diagram of wild-type or mutant F3 human ANGPTL2 reporter constructs (upper panel). Open and solid circles indicate wild-type and mutant putative E-boxes, respectively. Sequences of wild-type and mutant putative E-box sites are also shown (lower panel). Bold case letters indicate mutated sequences. B, Comparison of luciferase activity among HEK293 cells transiently co-transfected with indicated human ANGPTL2 reporters plus CLOCK and BMAL1 expression plasmids (n  =  4). Reporter activity of cells co-transfected with control vector (−) was set to 1. Data are expressed as means ± S.E.M. **p < 0.01.
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pone-0057921-g004: Non-canonical E-boxes mediate CLOCK/BMAL1-dependent induction of human ANGPTL2 promoter activity.A, Schematic diagram of wild-type or mutant F3 human ANGPTL2 reporter constructs (upper panel). Open and solid circles indicate wild-type and mutant putative E-boxes, respectively. Sequences of wild-type and mutant putative E-box sites are also shown (lower panel). Bold case letters indicate mutated sequences. B, Comparison of luciferase activity among HEK293 cells transiently co-transfected with indicated human ANGPTL2 reporters plus CLOCK and BMAL1 expression plasmids (n  =  4). Reporter activity of cells co-transfected with control vector (−) was set to 1. Data are expressed as means ± S.E.M. **p < 0.01.

Mentions: CLOCK and BMAL1 heterodimers positively regulate transcriptional targets through E-box sites (CACGTG or CACGTT) [1], [2], [5]. Since the −618 to −167 region of human ANGPTL2 promoter is necessary for CLOCK/BMAL1-dependent promoter activity, we searched for E-boxes in this region of the promoter. We identified four putative E-box sites (E1 at −393, E2 at −272, E3 at −262, and E4 at −197 from the human ANGPTL2 transcription start site, and all were non-canonical E-box motifs (Figure 4A). To investigate their potential function in CLOCK/BMAL1-dependent induction of ANGPTL2 promoter activity, we generated F3 constructs containing mutant E-box sites (mE1, mE2, mE3, or mE4) (Figure 4A) and co-transfected them into HEK293 cells along with CLOCK and BMAL1 expression plasmids (Figure 4B). We observed no difference in CLOCK/BMAL1-induced reporter activity between an F3 construct containing a mutant E1 site (F3-mE1) and a wild-type F3 construct. However, CLOCK/BMAL1-dependent reporter induction was significantly decreased when we employed constructs containing a mutant E2 (F3-mE2) or E4 (F3-mE4) site, while CLOCK/BMAL1-dependent reporter activity was only partially suppressed by the F3-mE3 mutation. CLOCK/BMAL1-induced activity of F3 constructs containing both mutant E2 and E3 sites (F3-mE2/3) or E3 and E4 sites (F3-mE3/4) was equivalent to that of F3-mE2 or F3-mE4 constructs, respectively. On the other hand, CLOCK/BMAL1-induced reporter activity of the F3 construct containing mutant E2 and E4 sites (F3-mE2/4) was significantly decreased compared to that seen with F3-mE2 or F3-mE4 constructs. These results suggest that the E2 and E4 sites of the human ANGPTL2 promoter are required for CLOCK/BMAL1-mediated transcriptional activation of ANGPTL2.


A molecular clock regulates angiopoietin-like protein 2 expression.

Kadomatsu T, Uragami S, Akashi M, Tsuchiya Y, Nakajima H, Nakashima Y, Endo M, Miyata K, Terada K, Todo T, Node K, Oike Y - PLoS ONE (2013)

Non-canonical E-boxes mediate CLOCK/BMAL1-dependent induction of human ANGPTL2 promoter activity.A, Schematic diagram of wild-type or mutant F3 human ANGPTL2 reporter constructs (upper panel). Open and solid circles indicate wild-type and mutant putative E-boxes, respectively. Sequences of wild-type and mutant putative E-box sites are also shown (lower panel). Bold case letters indicate mutated sequences. B, Comparison of luciferase activity among HEK293 cells transiently co-transfected with indicated human ANGPTL2 reporters plus CLOCK and BMAL1 expression plasmids (n  =  4). Reporter activity of cells co-transfected with control vector (−) was set to 1. Data are expressed as means ± S.E.M. **p < 0.01.
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pone-0057921-g004: Non-canonical E-boxes mediate CLOCK/BMAL1-dependent induction of human ANGPTL2 promoter activity.A, Schematic diagram of wild-type or mutant F3 human ANGPTL2 reporter constructs (upper panel). Open and solid circles indicate wild-type and mutant putative E-boxes, respectively. Sequences of wild-type and mutant putative E-box sites are also shown (lower panel). Bold case letters indicate mutated sequences. B, Comparison of luciferase activity among HEK293 cells transiently co-transfected with indicated human ANGPTL2 reporters plus CLOCK and BMAL1 expression plasmids (n  =  4). Reporter activity of cells co-transfected with control vector (−) was set to 1. Data are expressed as means ± S.E.M. **p < 0.01.
Mentions: CLOCK and BMAL1 heterodimers positively regulate transcriptional targets through E-box sites (CACGTG or CACGTT) [1], [2], [5]. Since the −618 to −167 region of human ANGPTL2 promoter is necessary for CLOCK/BMAL1-dependent promoter activity, we searched for E-boxes in this region of the promoter. We identified four putative E-box sites (E1 at −393, E2 at −272, E3 at −262, and E4 at −197 from the human ANGPTL2 transcription start site, and all were non-canonical E-box motifs (Figure 4A). To investigate their potential function in CLOCK/BMAL1-dependent induction of ANGPTL2 promoter activity, we generated F3 constructs containing mutant E-box sites (mE1, mE2, mE3, or mE4) (Figure 4A) and co-transfected them into HEK293 cells along with CLOCK and BMAL1 expression plasmids (Figure 4B). We observed no difference in CLOCK/BMAL1-induced reporter activity between an F3 construct containing a mutant E1 site (F3-mE1) and a wild-type F3 construct. However, CLOCK/BMAL1-dependent reporter induction was significantly decreased when we employed constructs containing a mutant E2 (F3-mE2) or E4 (F3-mE4) site, while CLOCK/BMAL1-dependent reporter activity was only partially suppressed by the F3-mE3 mutation. CLOCK/BMAL1-induced activity of F3 constructs containing both mutant E2 and E3 sites (F3-mE2/3) or E3 and E4 sites (F3-mE3/4) was equivalent to that of F3-mE2 or F3-mE4 constructs, respectively. On the other hand, CLOCK/BMAL1-induced reporter activity of the F3 construct containing mutant E2 and E4 sites (F3-mE2/4) was significantly decreased compared to that seen with F3-mE2 or F3-mE4 constructs. These results suggest that the E2 and E4 sites of the human ANGPTL2 promoter are required for CLOCK/BMAL1-mediated transcriptional activation of ANGPTL2.

Bottom Line: Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression.We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells.Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. tkado@gpo.kumamoto-u.ac.jp

ABSTRACT
Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

Show MeSH
Related in: MedlinePlus