HBP1 Repression of the p47phox Gene: Cell Cycle Regulation via the NADPH Oxidase

Abstract

Several studies have linked the production of reactive oxygen species (ROS) by the NADPH oxidase to cellular growth control. In many cases, activation of the NADPH oxidase and subsequent ROS generation is required for growth factor signaling and mitogenesis in nonimmune cells. In this study, we demonstrate that the transcriptional repressor HBP1 (HMG box-containing protein 1) regulates the gene for the p47phox regulatory subunit of the NADPH oxidase. HBP1 represses growth regulatory genes (e.g., N-Myc, c-Myc, and cyclin D1) and is an inhibitor of G₁ progression. The promoter of the p47phox gene contains six tandem high-affinity HBP1 DNA-binding elements at positions −1243 to −1318 bp from the transcriptional start site which were required for repression. Furthermore, HBP1 repressed the expression of the endogenous p47phox gene through sequence-specific binding. With HBP1 expression and the subsequent reduction in p47phox gene expression, intracellular superoxide production was correspondingly reduced. Using both the wild type and a dominant-negative mutant of HBP1, we demonstrated that the repression of superoxide production through the NADPH oxidase contributed to the observed cell cycle inhibition by HBP1. Together, these results indicate that HBP1 may contribute to the regulation of NADPH oxidase-dependent superoxide production through transcriptional repression of the p47phox gene. This study defines a transcriptional mechanism for regulating intracellular ROS levels and has implications in cell cycle regulation.

We thank Robert A. Clark for the p47phox promoter constructs and Tom Leto for the p47phox antisera. We also thank Peggy Farnham for generous access to her detailed ChIP protocol.

The support of the GRASP Digestive Disease Center at New England Medical Center (grant P30 DK34928) and its Molecular Biology/Genomics Core is gratefully acknowledged. This work was supported by grants to A.S.Y. from the U.S. Army (grant BC990538) and from the NIH (grants GM44634 and CA94187) and to K.E.P. from the NIH (grant ES11518).