Liu Yi, Zhang Yingjia, Schmelzer Kara, Lee Tzong-Shyuan, Fang Xiang, Zhu Yi, Spector Arthur A, Gill Sarjeet, Morisseau Christophe, Hammock Bruce D, Shyy John Y-J
Division of Biomedical Sciences, University of California, Riverside, CA 92521, USA.
Proc Natl Acad Sci U S A. 2005 Nov 15;102(46):16747-52. doi: 10.1073/pnas.0508081102. Epub 2005 Nov 2.
We previously reported that laminar flow activates peroxisome proliferator-activated receptor gamma (PPARgamma) in vascular endothelial cells in a ligand-dependent manner that involves phospholipase A2 and cytochrome P450 epoxygenases. In this study, we investigated whether epoxyeicosatrienoic acids (EETs), the catalytic products of cytochrome P450 epoxygenases, are PPARgamma ligands. Competition and direct binding assays revealed that EETs bind to the ligand-binding domain of PPARgamma with K(d) in the microM range. In the presence of adamantyl-ureido-dodecanoic acid (AUDA), a soluble epoxide hydrolase (sEH)-specific inhibitor, EETs increased PPARgamma transcription activity in endothelial cells and 3T3-L1 preadipocytes. Inclusion of AUDA in the perfusing media enhanced, but overexpression of sEH reduced, the laminar flow-induced PPARgamma activity. Furthermore, laminar flow augmented cellular levels of EETs but decreased sEH at the levels of mRNA, protein, and activity. Blocking PPARgamma by GW9662 abolished the EET/AUDA-mediated antiinflammatory effect, which indicates that PPARgamma is an effector of EETs.
我们之前报道过，层流以一种依赖配体的方式激活血管内皮细胞中的过氧化物酶体增殖物激活受体γ（PPARγ），这种方式涉及磷脂酶A2和细胞色素P450环氧合酶。在本研究中，我们调查了细胞色素P450环氧合酶的催化产物环氧二十碳三烯酸（EETs）是否为PPARγ配体。竞争和直接结合试验表明，EETs以微摩尔范围内的解离常数（K(d)）与PPARγ的配体结合域结合。在存在金刚烷基脲基十二烷酸（AUDA）（一种可溶性环氧化物水解酶（sEH）特异性抑制剂）的情况下，EETs增加了内皮细胞和3T3-L1前脂肪细胞中的PPARγ转录活性。在灌注培养基中加入AUDA可增强层流诱导的PPARγ活性，但sEH的过表达则降低该活性。此外，层流增加了细胞内EETs的水平，但在mRNA、蛋白质和活性水平上降低了sEH。用GW9662阻断PPARγ消除了EET/AUDA介导的抗炎作用，这表明PPARγ是EETs的效应器。
