Human ACCATTTATAAC AGAGGTGAACAT ACAAG CCTGATATAACA TCACAGTAGACA TGAAAA TGTATGCCATCAG ACCCAGCAACTCT CA Angptl 3 Mouse GATTTGCTATGT TGGATGATGTCAA CTTATGGACAAAA TCTTTAAGTCCA TGA ATTTTAGCGAATG GCCTCCTGCAGCT CYP 7 A Mouse CAAAACCTCCAA TCTGTCATGAGA GCGTTAGATATC CGGCTTCAAA

Angiopoietins are members of the vascular endothelial growth factor family. One family member, angiopoietin-like protein 3 (Angptl3), was recently shown to be predominantly expressed in the liver and to play an important role in regulating lipid metabolism. In this study, we show that the Angptl3 gene is a direct target of the liver X receptor (LXR). Mice fed a high cholesterol diet exhibited a significant increase in Angptl3 expression in the liver. Oral administration to mice of T0901317, a synthetic LXR-selective agonist, increases levels of plasma lipids and Angptl3 mRNA in the liver. Treatment of HepG2 cells with LXR selective agonists led to a dose-dependent increase of Angptl3 mRNA. Analysis of the DNA sequence just 5 of the Angptl3 transcriptional start site revealed the presence of several potential transcription factor binding sites, including that for LXR. When transfected into HepG2 cells, the promoter activity of Angptl3 was significantly induced by LXRor retinoid X receptor-selective agonists. Mutation of the predicted LXR binding site (DR4 element) completely abolished the LXR agonistmediated activation of the promoter. Together, these studies show that Angptl3 is transcriptionally regulated by LXR, and reveals a novel mechanism by which LXR may regulate lipid metabolism. — Kaplan, R. , T. Zhang, M. Hernandez, F. X. Gan, S. D. Wright, M. G. Waters, and T-Q. Cai. Regulation of the angiopoietin-like protein 3gene by LXR. J. Lipid Res. 2003. 44: 136–143. Supplementary key words lipid metabolism • angiopoietin • transcriptional regulation • nuclear receptor The angiopoietins are a family of secreted growth factors. Together with their respective endothelium-specific surface receptors, such as the receptor tyrosine kinase Tie2, the angiopoietins play important roles in angiogenesis [as reviewed in ref. (1)]. One family member, Angiopoietin-like protein 3 (Angptl3), was previously found to be expressed in a liver specific manner (2), and more recently, it was found to play an important role in the regulation of lipid metabolism (3). Mutation of Angptl3 results in low levels of plasma lipids in KK/San mice (3). These hypolipidemic mice are derived from KK obese mice, which display a multigenic syndrome of moderate obesity and a diabetic phenotype including hyperinsulinemia, hyperglycemia, and hyperlipidemia (4–6). Administration or overexpression of Angptl3 in mice elicited a rapid increase in circulating levels of plasma cholesterol, triglycerides (TG), and non-esterified fatty acids (3). While these studies revealed a role of Angptl3 in lipid metabolism, they did not address the molecular mechanism by which a hyperlipidemic response was mediated by Angptl3. Understanding the regulation of Angptl3 expression is an important step toward a better comprehension of the physiological network that comprises lipid metabolism. Many genes involved in lipid metabolism are regulated by nuclear receptors. Liver X receptors (LXRs), including LXR and LXR , are members of the nuclear receptor superfamily [as reviewed in ref. (7)]. LXRs heterodimerize with retinoid X receptors (RXRs) and can be activated by both RXR and LXR ligands. Upon activation, LXRs regulate the expression of target genes by binding to specific promoter response elements (termed LXREs) that contain a hexameric nucleotide direct repeat separated by four bases (DR4). Recent studies have shown that these ligand-activated transcription factors play important roles in the regulation of genes that govern cholesterol homeostasis in the liver and peripheral tissues (7). In addition to cholesterol metabolism, accumulating evidence suggests that LXRs also play important roles in regulating fatty acid metabolism. For example, the expressions of sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase, and lipoprotein lipase were all shown to be strongly activated by LXR agonists (8–10). It has been suggested that the regulation of SREBP-1c by LXR may contribute to increased levels of plasma TG when animals are treated with LXR agonists (11–12). However, SREBP-1c cannot be solely responsible for the elevated TG because mice overexpressing SREBP-1c had a reduced level of circulating TG (13). To determine if Angptl3 might be such a factor, we studAbbreviations: ABCA1, ATP-binding cassette transporter 1; Angptl3, angiopoietin-like protein 3; FCS, fetal calf serum; LXR, liver X receptor; OH Ch, hydroxycholesterol; 9-RA, 9-cis retinoic acid; SREBP, sterol regulatory element-binding protein; TG, triglycerides. 1 To whom correspondence should be addressed. e-mail: tianquan_cai@merck.com Manuscript received 16 September 2002. Published, JLR Papers in Press, October 16, 2002. DOI 10.1194/jlr.M200367-JLR200 by gest, on A uust 8, 2017 w w w . j.org D ow nladed fom