Laniado-Schwartzman M, Lavrovsky Y, Stoltz R A, Conners M S, Falck J R, Chauhan K, Abraham N G
Department of Pharmacology, New York Medical College, Valhalla 10595.
J Biol Chem. 1994 Sep 30;269(39):24321-7.
12(R)-Hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid (12(R)-HETrE) is an arachidonic acid metabolite formed by the corneal epithelium of several species, porcine leukocytes, and human and rat epidermal cells. It is a potent, stereospecific proinflammatory and angiogenic factor and its synthesis is increased manyfold in inflamed tissues, e.g. cornea and skin. It is possible that the angiogenic activity of 12(R)-HETrE is due to a direct mitogenic effect on microvessel endothelial cells via yet to be elucidated cellular and molecular mechanisms. In the present study, we demonstrated the ability of 12(R)-HETrE to stimulate the growth of quiescent endothelial cells in a time- and concentration-dependent manner with a maximal effect at 0.1 nM. This effect was highly stereospecific since its enantiomer, 12(S)-HETrE, had no effect within the same concentration range. Northern blot analysis and transient transfection experiments with chloramphenicol acetyltransferase constructs of oncogene promoter regions demonstrated significant increases over control (0.5% fetal calf serum) in c-myc-, c-jun, and c-fos mRNA levels and expression in cells treated with 0.1 nM 12(R)-HETrE. Electrophoretic mobility shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE with specific radiolabeled oligonucleotides corresponding to known transcriptional binding sites, including AP-1, AP-2, SP1, TRE, NF kappa B, TFIID, OKT1, CREB, CTF/NF1, and GRE demonstrated a markedly rapid and specific increase in the binding activity of NF kappa B and to a lesser extent, AP-1. No significant increase was observed in the binding of other transcription factors assayed as compared to control (untreated) cells. Since the protooncogenes (c-fos, c-jun, and c-myc) are immediate early response genes that are implicated in the process of cell proliferation and differentiation, and activation of certain transcription factors, in particular NF kappa B, is associated with the immediate response of the cell to an injury, we propose that 12(R)HETrE's mitogenic and angiogenic activities are mediated, in part, via the activation of NF kappa B and expression of these protooncogenes.
12(R)-羟基-5,8,14(Z,Z,Z)-二十碳三烯酸（12(R)-HETrE）是一种由多种物种的角膜上皮、猪白细胞以及人和大鼠表皮细胞形成的花生四烯酸代谢产物。它是一种强效的、立体特异性的促炎和血管生成因子，在炎症组织如角膜和皮肤中其合成会增加许多倍。12(R)-HETrE的血管生成活性可能是由于其通过尚未阐明的细胞和分子机制对微血管内皮细胞产生直接的促有丝分裂作用。在本研究中，我们证明了12(R)-HETrE能够以时间和浓度依赖性方式刺激静止内皮细胞的生长，在0.1 nM时具有最大效应。这种效应具有高度的立体特异性，因为其对映体12(S)-HETrE在相同浓度范围内没有作用。用癌基因启动子区域的氯霉素乙酰转移酶构建体进行的Northern印迹分析和瞬时转染实验表明，在用0.1 nM 12(R)-HETrE处理的细胞中，c-myc、c-jun和c-fos mRNA水平及表达相对于对照（0.5%胎牛血清）有显著增加。用对应于已知转录结合位点（包括AP-1、AP-2、SP1、TRE、NFκB、TFIID、OKT1、CREB、CTF/NF1和GRE）的特异性放射性标记寡核苷酸对用12(R)-HETrE处理的细胞的核蛋白提取物进行电泳迁移率变动分析，结果显示NFκB的结合活性显著快速且特异性增加，AP-1的结合活性增加程度较小。与对照（未处理）细胞相比，在检测的其他转录因子的结合中未观察到显著增加。由于原癌基因（c-fos、c-jun和c-myc）是与细胞增殖和分化过程相关的即刻早期反应基因，并且某些转录因子（特别是NFκB）的激活与细胞对损伤的即刻反应相关，我们提出12(R)-HETrE的促有丝分裂和血管生成活性部分是通过NFκB的激活和这些原癌基因的表达介导的。
