Department of Chemical and Systems Biology, Stanford University School of Medicine Clark Center W200, Stanford, CA, USA.
Oncogene. 2010 Aug 5;29(31):4473-84. doi: 10.1038/onc.2010.200. Epub 2010 Jun 7.
Reactive oxygen species (ROS) are produced in growth factor-signaling pathways leading to cell proliferation, but the mechanisms leading to ROS generation and the targets of ROS signals are not well understood. Using a focused siRNA screen to identify redox-related proteins required for growth factor-induced cell cycle entry, we show that two ROS-generating proteins, the NADPH oxidases NOX4 and DUOX2, are required for platelet-derived growth factor (PDGF) induced retinoblastoma protein (Rb) phosphorylation in normal human fibroblasts. Unexpectedly, NOX4 and DUOX2 knockdown did not inhibit the early signaling pathways leading to cyclin D1 upregulation. However, hours after growth factor stimulation, NOX4 and DUOX2 knockdown reduced ERK1 phosphorylation and increased levels of the tumor suppressor protein p53 and a cell cycle inhibitor protein p21 (Waf1/Cip1) that is transcriptionally regulated by p53. Co-knockdown of NOX4 or DUOX2 with either p53 or with p21 overcame the inhibition of Rb phosphorylation that occurred with NOX4 or DUOX2 knockdown alone. Our results argue that rather than primarily affecting growth factor receptor signaling, NOX4 and DUOX2 regulate cell cycle entry as part of a p53-dependent checkpoint for proliferation.
活性氧（ROS）在生长因子信号通路中产生，导致细胞增殖，但ROS 产生的机制和 ROS 信号的靶标尚不清楚。我们使用有针对性的 siRNA 筛选来鉴定与生长因子诱导的细胞周期进入相关的氧化还原相关蛋白，结果表明，两种产生 ROS 的蛋白，即 NADPH 氧化酶 NOX4 和 DUOX2，对于血小板衍生生长因子（PDGF）诱导的正常人类成纤维细胞中视网膜母细胞瘤蛋白（Rb）磷酸化是必需的。出乎意料的是，NOX4 和 DUOX2 的敲低并不抑制导致细胞周期蛋白 D1 上调的早期信号通路。然而，在生长因子刺激数小时后，NOX4 和 DUOX2 的敲低减少了 ERK1 磷酸化，并增加了肿瘤抑制蛋白 p53 和细胞周期抑制剂蛋白 p21（Waf1/Cip1）的水平，p21 可被 p53 转录调控。NOX4 或 DUOX2 与 p53 或 p21 的共敲低克服了单独敲低 NOX4 或 DUOX2 时发生的 Rb 磷酸化抑制。我们的结果表明，NOX4 和 DUOX2 调节细胞周期进入，作为增殖的 p53 依赖性检查点的一部分，而不是主要影响生长因子受体信号。
