Tumor necrosis factor α activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells

Abstract NADPH oxidase 1 (Nox1) is a multicomponent enzyme consisting of p22 phox , Nox organizer 1 (NOXO1), Nox1 activator 1, and Rac1. Interleukin-1β, flagellin, interferon-γ, and tumor necrosis factor α (TNF-α) similarly induced Nox1 in a colon cancer cell line (T84), whereas only TNF-α fully induced NOXO1 and upregulated superoxide-producing activity by ninefold. This upregulation was canceled by knockdown of NOXO1 with small interfering RNAs. TNF-α rapidly phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase 1/2, followed by phosphorylation of c-Jun and c-Fos and appearance of an AP-1 binding activity within 30 min. We cloned the 5′ flank of the human NOXO1 gene (– 3888 to + 263 bp), and found that the region between – 585 and – 452 bp, which contains consensus elements of YY-1, AP-1, and Ets, and the GC-rich region encoding three putative binding sites for SP-1, was crucial for TNF-α-dependent promoter activity. Serial mutation analysis of the elements identified an AP-1 binding site (from – 561 to – 551 bp, agtAAGtcatg ) as a crucial element for TNF-α-stimulated transcription of the human NOXO1 gene, which was also confirmed by the AP-1 decoy experiments. Thus, TNF-α acts as a potent activator of Nox1-based oxidase in colon epithelial cells, suggesting a potential role of this oxidase in inflammation of the colon.