Sekharam M, Cunnick J M, Wu J
Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612, USA.
Biochem J. 2000 Mar 15;346 Pt 3(Pt 3):751-8.
Although it is now recognized that low levels of reactive oxygen species (ROS) are required for the mitogenic response, mitogen-induced signalling pathways that regulate ROS generation in non-phagocytic cells remain largely uncharacterized. Using a real-time assay for measuring hydrogen peroxide (H(2)O(2)) formation, we analysed H(2)O(2) release in human HaCaT keratinocytes in response to lysophosphatidic acid (LPA), a mitogen for keratinocytes. LPA rapidly increased H(2)O(2) release in HaCaT cells. Unlike LPA-induced mitogen-activated protein (MAP) kinase activation, LPA-stimulated H(2)O(2) release was independent of the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Calcium chelators, phospholipase A(2) inhibitors, and lipoxygenase inhibitors effectively blocked LPA-stimulated H(2)O(2) release, whereas cyclooxygenase inhibitors were without effect. Addition of 5-lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B(4), but not 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C(4), restored LPA-stimulated H(2)O(2) release in cells treated with the lipoxygenase inhibitors nordihydroguaiaretic acid and Zileuton. These results suggest that the lipoxygenase products 5-HPETE and leukotriene B(4) are required for LPA-stimulated H(2)O(2) release in HaCaT cells.
尽管现在人们认识到有丝分裂原反应需要低水平的活性氧（ROS），但在非吞噬细胞中调节ROS生成的有丝分裂原诱导信号通路在很大程度上仍未得到充分表征。我们使用一种实时检测方法来测量过氧化氢（H₂O₂）的形成，分析了人HaCaT角质形成细胞中响应溶血磷脂酸（LPA，一种角质形成细胞的有丝分裂原）时H₂O₂的释放情况。LPA迅速增加了HaCaT细胞中H₂O₂的释放。与LPA诱导的丝裂原活化蛋白（MAP）激酶激活不同，LPA刺激的H₂O₂释放不依赖于表皮生长因子（EGF）受体的酪氨酸激酶活性。钙螯合剂、磷脂酶A₂抑制剂和脂氧合酶抑制剂可有效阻断LPA刺激的H₂O₂释放，而环氧化酶抑制剂则无效。添加5-脂氧合酶产物5-氢过氧化二十碳四烯酸（5-HPETE）和白三烯B₄，但不添加5-羟基二十碳四烯酸（5-HETE）和白三烯C₄，可恢复用脂氧合酶抑制剂去甲二氢愈创木酸和齐留通处理的细胞中LPA刺激的H₂O₂释放。这些结果表明，脂氧合酶产物5-HPETE和白三烯B₄是HaCaT细胞中LPA刺激H₂O₂释放所必需的。
