A Molecular Clock Regulates Angiopoietin-Like Protein 2 Expression

<div><p>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 <i>ANGPTL2</i> expression are poorly understood. Here, we assess circadian rhythmicity of <i>ANGPTL2</i> expression in various mouse tissues. We observed that <i>ANGPTL2</i> rhythmicity was similar to that of the <i>PER2</i> gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human <i>ANGPTL2</i> gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the <i>ANGPTL2</i> promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, <i>Cry</i>-deficient mice exhibited arrhythmic <i>Angptl2</i> expression. Taken together, these data suggest that periodic expression of <i>ANGPTL2</i> is regulated by a molecular clock.</p> </div

Periodicity of <i>Angptl2</i> mRNA and protein expression in epididymal WAT.

<p>A and B, Temporal expression profiles of <i>Angptl2</i> mRNA in epididymal WAT of mice housed under light/dark cycles (<b>A</b>) or under constant darkness (<b>B</b>). C, Upper panel: Representative image of immunoblotting of ANGPTL2 protein in epididymal WAT. Hsc70 served as a control. Lower panel: Quantification of ANGPTL2 protein levels relative to Hsc70. For mice housed under constant darkness, circadian time (CT) was used instead of Zeitgeber time. The average expression level of mRNA or protein across all time points was set to 1. Data are expressed as means ± S.E.M. (n  =  3–5 mice for each data point).</p

Endogenous CLOCK binds to E-boxes of the human <i>ANGPTL2</i> promoter in U2OS cells.

<p>A, Upper panel: Schematic diagram of the location of primers flanking E-box sites of the human ANGPTL2 promoter. Arrowheads indicate specific primers. Lower panel: Representative image of ChIP assays showing CLOCK binding to E-box sites of <i>ANGPTL2</i> and <i>PER2</i> promoters in unsynchronized U2OS cells. Purified DNA fragments derived from immunoprecipitated chromatin complexes were analyzed by PCR using primers specific for the E-box sites of <i>ANGPTL2</i> and <i>PER2</i> promoters or for the <i>GAPDH</i> promoter (negative control). B, Representative image of ChIP assays showing oscillatory CLOCK binding to E-boxes of the human <i>ANGPTL2</i> promoter in U2OS cells after serum shock. Chromatin was collected at 16 h and 28 h after serum shock and subjected to ChIP assays of the human <i>ANGPTL2</i> promoter or of the <i>GAPDH</i> promoter (negative control). Each experiment was performed at least three times.</p

<i>Cry</i>-deficient mice show arrhythmic <i>Angptl2</i> expression.

<p>Relative levels of <i>Angptl2</i>, <i>Per2</i>, and <i>Rev-erbα</i> mRNA expression in WAT of <i>Cry</i>-deficient or wild-type mice at CT 2 and CT 12 (n  =  4 for each data point). The expression level in wild-type mice at CT 12 was set to 100%. Data are expressed as means ± S.E.M. **<i>p</i> < 0.01. n.s., no statistical difference.</p

Co-expression of CLOCK and BMAL1 activates human <i>ANGPTL2</i> promoter activity.

<p>A, Upper panel: Schematic diagram of human <i>ANGPTL2</i> reporter constructs. Gray box indicates the <i>luciferase</i> 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 <i>ANGPTL2</i> 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 <i>ANGPTL2</i> reporters plus CLOCK and BMAL1 expression plasmids (n  =  4). <i>Per1</i> and <i>Per2</i> 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 <i>ANGPTL2</i> 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. **<i>p</i> < 0.01.</p

Temporal expression of <i>Angptl2</i> and core clock genes in mouse epididymal white adipose tissue.

<p>Temporal expression of <i>Angptl2</i> (<b>A</b>), <i>Clock</i> (<b>B</b>), <i>Bmal1</i> (<b>C</b>), <i>Cry1</i> (<b>D</b>), <i>Per2</i> (<b>E</b>), <i>Rev-erbα</i> (<b>F</b>), and <i>Rorα</i> (<b>G</b>) mRNA in epididymal white adipose tissue (WAT) of mice housed under light/dark cycles. For light/dark cycles, Zeitgeber time (ZT) 0 or ZT 24 was designated as lights on and ZT 12 or ZT 36 as lights off. The average mRNA expression level across all time points was set to 1. Data are expressed as means ± S.E.M. (n  =  3 mice for each data point).</p