Nony Paul A, Kennett Sarah B, Glasgow Wayne C, Olden Kenneth, Roberts John D
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 2005 Sep 9;280(36):31413-9. doi: 10.1074/jbc.M500418200. Epub 2005 Jul 6.
The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.
膳食中的顺式多不饱和脂肪酸花生四烯酸可刺激转移性人乳腺癌细胞（MDA-MB-435）与细胞外基质的黏附，但脂肪酸改变这些细胞行为的分子机制尚不清楚。暴露于花生四烯酸会激活多种信号通路。p38丝裂原活化蛋白激酶（p38 MAPK）的激活是细胞对IV型胶原黏附增加所必需的，且这种激活对脂氧合酶抑制剂敏感，提示花生四烯酸代谢是必需的。本研究的目的是确定一种或多种负责激活p38 MAPK的花生四烯酸关键代谢产物，并阐明导致p38 MAPK激活的上游激酶。高效液相色谱分析显示，MDA-MB-435细胞将外源性花生四烯酸主要代谢为15（S）-羟基二十碳四烯酸（15（S）-HETE）。用针对15（S）-脂氧合酶-1（LOX-1）和15（S）-脂氧合酶-2（LOX-2）的特异性抗体进行免疫印迹分析表明，这些肿瘤细胞中表达15-LOX-2，而不表达15-LOX-1。脂氧合酶抑制剂去甲二氢愈创木酸可减弱15（S）-HETE的产生，并抑制暴露于花生四烯酸后p38 MAPK的磷酸化。相反，LOX-2的过表达使细胞对添加花生四烯酸敏感，导致p38 MAPK的激活增加。向MDA-MB-435细胞中添加外源性15（S）-HETE可刺激细胞对IV型胶原的黏附，并激活p38 MAPK途径，包括上游激酶转化生长因子-β1激活蛋白激酶-1（TAK1）和丝裂原活化蛋白激酶激酶6。用显性负性形式的TAK1转染这些细胞可阻断花生四烯酸刺激的p38 MAPK磷酸化。这些数据表明，15（S）-LOX-2生成的15（S）-HETE激活特定的生长因子受体相关信号通路，从而启动导致细胞与细胞外基质黏附增加的信号转导事件。
