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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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Co-expression of CLOCK and BMAL1 activates human ANGPTL2 promoter activity.A, Upper panel: Schematic diagram of human ANGPTL2 reporter constructs. Gray box indicates the luciferase gene. The number of nucleotide residues indicates the distance from the putative transcription start site (+1). Lower panel: Comparison of luciferase activity among HEK293 cells transiently transfected with indicated human ANGPTL2 reporters or with pGL3-Basic (Basic) (n  =  3). Reporter activity of cells transfected with pGL3-Basic was set to 1. B, Comparison of luciferase activity of HEK293 cells transiently co-transfected with indicated human ANGPTL2 reporters plus CLOCK and BMAL1 expression plasmids (n  =  4). Per1 and Per2 reporters served as positive controls. Reporter activity of cells co-transfected with control vector (−) was set to 1. C, Comparison of luciferase activity among HEK293 cells transiently co-transfected with the F3 human ANGPTL2 reporter plus CLOCK and BMAL1 expression plasmids, with or without a CRY expression plasmid (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-g003: Co-expression of CLOCK and BMAL1 activates human ANGPTL2 promoter activity.A, Upper panel: Schematic diagram of human ANGPTL2 reporter constructs. Gray box indicates the luciferase gene. The number of nucleotide residues indicates the distance from the putative transcription start site (+1). Lower panel: Comparison of luciferase activity among HEK293 cells transiently transfected with indicated human ANGPTL2 reporters or with pGL3-Basic (Basic) (n  =  3). Reporter activity of cells transfected with pGL3-Basic was set to 1. B, Comparison of luciferase activity of HEK293 cells transiently co-transfected with indicated human ANGPTL2 reporters plus CLOCK and BMAL1 expression plasmids (n  =  4). Per1 and Per2 reporters served as positive controls. Reporter activity of cells co-transfected with control vector (−) was set to 1. C, Comparison of luciferase activity among HEK293 cells transiently co-transfected with the F3 human ANGPTL2 reporter plus CLOCK and BMAL1 expression plasmids, with or without a CRY expression plasmid (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: Angptl2 mRNA expression showed circadian periodicity similar to that of Per2 (Figure 1A and E). Since PER2 circadian expression is regulated by the CLOCK/BMAL1 complex [1], we hypothesized that CLOCK and BMAL1 may regulate rhythmic ANGPTL2 expression. To assess molecular mechanisms underlying circadian ANGPTL2 expression, we performed luciferase assays in HEK293 cells using human ANGPTL2 reporter constructs. Recently, we reported that the F4 construct (containing −168 to +98 of human ANGPTL2) exhibits very high reporter activity in human lung cancer cells [13]. Similarly, in HEK293 cells, we confirmed that the F4 construct shows very high reporter activity, while the activity of the F5 construct (containing −21 to +98) was significantly decreased (Figure 3A), indicating that the −168 to −22 region constitutes an essential minimal promoter.


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)

Co-expression of CLOCK and BMAL1 activates human ANGPTL2 promoter activity.A, Upper panel: Schematic diagram of human ANGPTL2 reporter constructs. Gray box indicates the luciferase gene. The number of nucleotide residues indicates the distance from the putative transcription start site (+1). Lower panel: Comparison of luciferase activity among HEK293 cells transiently transfected with indicated human ANGPTL2 reporters or with pGL3-Basic (Basic) (n  =  3). Reporter activity of cells transfected with pGL3-Basic was set to 1. B, Comparison of luciferase activity of HEK293 cells transiently co-transfected with indicated human ANGPTL2 reporters plus CLOCK and BMAL1 expression plasmids (n  =  4). Per1 and Per2 reporters served as positive controls. Reporter activity of cells co-transfected with control vector (−) was set to 1. C, Comparison of luciferase activity among HEK293 cells transiently co-transfected with the F3 human ANGPTL2 reporter plus CLOCK and BMAL1 expression plasmids, with or without a CRY expression plasmid (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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585275&req=5

pone-0057921-g003: Co-expression of CLOCK and BMAL1 activates human ANGPTL2 promoter activity.A, Upper panel: Schematic diagram of human ANGPTL2 reporter constructs. Gray box indicates the luciferase gene. The number of nucleotide residues indicates the distance from the putative transcription start site (+1). Lower panel: Comparison of luciferase activity among HEK293 cells transiently transfected with indicated human ANGPTL2 reporters or with pGL3-Basic (Basic) (n  =  3). Reporter activity of cells transfected with pGL3-Basic was set to 1. B, Comparison of luciferase activity of HEK293 cells transiently co-transfected with indicated human ANGPTL2 reporters plus CLOCK and BMAL1 expression plasmids (n  =  4). Per1 and Per2 reporters served as positive controls. Reporter activity of cells co-transfected with control vector (−) was set to 1. C, Comparison of luciferase activity among HEK293 cells transiently co-transfected with the F3 human ANGPTL2 reporter plus CLOCK and BMAL1 expression plasmids, with or without a CRY expression plasmid (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: Angptl2 mRNA expression showed circadian periodicity similar to that of Per2 (Figure 1A and E). Since PER2 circadian expression is regulated by the CLOCK/BMAL1 complex [1], we hypothesized that CLOCK and BMAL1 may regulate rhythmic ANGPTL2 expression. To assess molecular mechanisms underlying circadian ANGPTL2 expression, we performed luciferase assays in HEK293 cells using human ANGPTL2 reporter constructs. Recently, we reported that the F4 construct (containing −168 to +98 of human ANGPTL2) exhibits very high reporter activity in human lung cancer cells [13]. Similarly, in HEK293 cells, we confirmed that the F4 construct shows very high reporter activity, while the activity of the F5 construct (containing −21 to +98) was significantly decreased (Figure 3A), indicating that the −168 to −22 region constitutes an essential minimal promoter.

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