The NADPH oxidases NOX4 and DUOX2 regulate cell cycle entry via a p53-dependent pathway

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.