Raspé E, Duez H, Gervois P, Fiévet C, Fruchart J C, Besnard S, Mariani J, Tedgui A, Staels B
U325 INSERM, Institut Pasteur de Lille, 1 rue Calmette, 59019 Lille, France.
J Biol Chem. 2001 Jan 26;276(4):2865-71. doi: 10.1074/jbc.M004982200. Epub 2000 Oct 26.
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.
富含甘油三酯的残余脂蛋白被认为是动脉粥样硬化发病机制的主要危险因素。由于载脂蛋白（apo）C-III是血浆甘油三酯和残余脂蛋白代谢的主要决定因素，了解该基因的表达调控方式很重要。在本研究中，我们确定孤儿核受体RORα1是人及小鼠apo C-III基因表达的调节因子。RORα基因缺失的纯合 staggerer（sg/sg）小鼠的血浆甘油三酯和apo C-III蛋白浓度显著低于野生型同窝小鼠。sg/sg小鼠血浆apo C-III水平降低与肝脏和肠道中apo C-III mRNA水平降低有关。在人肝癌HepG2细胞、人结肠CaCO2细胞和兔肾RK13细胞中进行的瞬时转染实验表明，人RORα1亚型的过表达特异性增加人apo C-III启动子活性，表明RORα1增强人apo C-III基因转录。通过启动子缺失分析和凝胶迁移实验将RORα1反应元件定位到人apo C-III启动子中位于-83/-78（在C3P位点内）和-23/-18（TATA框下游）位置的两个AGGTCA半位点，其中-23/-18位点表现出最高的结合亲和力。在HepG2细胞中转染定点突变构建体表明，RORα1的作用主要由-23/-18位点介导。该位点在小鼠apo C-III基因启动子中保守。此外，RORα与小鼠的等效位点结合并激活含有克隆在异源启动子前的三个小鼠位点拷贝的构建体。综上所述，我们的数据确定RORα是apo C-III基因表达的转录调节因子，为RORα1在控制甘油三酯代谢的基因调控中提供了一种新的生理作用。
