Abstract
Triglyceride-rich remnant lipoproteins are considered as major risk factors contributing to the pathogenesis of atherosclerosis. Because apolipoprotein (apo) C-III is a major determinant of plasma triglyceride and remnant lipoprotein metabolism, it is important to understand how the expression of this gene is regulated. In the present study, we identified the orphan nuclear receptor RORalpha1 as a regulator of human and mouse apo C-III gene expression. Plasma triglyceride and apo C-III protein concentrations in staggerer (sg/sg) mice, homozygous for a deletion in the RORalpha gene, were significantly lower than in wild type littermates. The lowered plasma apo C-III levels were associated with reduced apo C-III mRNA levels in liver and intestine of sg/sg mice. Transient transfection experiments in human hepatoma HepG2, human colonic CaCO2, and rabbit kidney RK13 cells demonstrated that overexpression of the human RORalpha1 isoform specifically increases human apo C-III promoter activity, indicating that RORalpha1 enhances human apo C-III gene transcription. RORalpha1 response elements were mapped by promoter deletion analysis and gel shift experiments to two AGGTCA half-sites located at positions -83/-78 (within the C3P site) and -23/-18 (downstream of the TATA box) in the human apo C-III promoter, with the -23/-18 site exhibiting the highest binding affinity. Transfection of site-directed mutated constructs in HepG2 cells indicated that the RORalpha1 effect is predominantly mediated by the -23/-18 site. This site is conserved in the mouse apo C-III gene promoter. Moreover, RORalpha binds to the equivalent mouse site and activates constructs containing three copies of the mouse site cloned in front of an heterologous promoter. Taken together, our data identify RORalpha as a transcriptional regulator of apo C-III gene expression, providing a novel, physiological role for RORalpha1 in the regulation of genes controlling triglyceride metabolism.
Highlights
Triglyceride-rich remnant lipoproteins are considered as major risk factors contributing to the pathogenesis of atherosclerosis
Apolipoprotein1 C-III is a 79-amino acid glycoprotein synthesized in the liver and, to a lesser extent, in the intestine, that plays a key role in plasma triglyceride metabolism as evidenced by pharmacological [7,8,9], clinical [10, 11], genetic [12], and experimental data in transgenic animal models [13]
Plasma Triglyceride and apo C-III Concentrations as Well as Hepatic and Intestinal apo C-III mRNA Levels Are Decreased in staggerer Mice—To determine whether staggerer mice display altered triglyceride metabolism, plasma triglycerides were measured in overnight fasted female staggerer mice and compared with age-matched wild type C57BL/6 littermates (Fig. 1)
Summary
Triglyceride-rich remnant lipoproteins are considered as major risk factors contributing to the pathogenesis of atherosclerosis. Apo C-III deficiency in humans [16] or apo C-III gene disruption in transgenic mice [17] results in increased catabolism of very low density lipoprotein particles, whereas increased apo C-III synthesis occurs in hypertriglyceridemic patients [18] Results from both in vivo (19 – 22) and in vitro [23,24,25] studies indicate that apo C-III delays the catabolism of triglyceride-rich particles. The C3P site located at position Ϫ87/Ϫ67 relative to the transcription start site is a major determinant of apo C-III promoter activity [35, 36] It contains a direct repeat of two AGGTCA half-sites separated by one nucleotide (DR-1) to which the nuclear hormone receptors HNF-4, PPAR, RXR, Ear, COUPTF-I, and COUPTF-II are binding [8, 9, 37, 39, 40]. T3R [41], ATF-2 [42], NFB [43], and Jun [42] regulatory elements have been identified in the human apo C-III promoter
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