Identification of NF-κB-regulated genes induced by TNFα utilizing expression profiling and RNA interference

Abstract

Tumor necrosis factor (TNF) is a proinflammatory cytokine with important roles in regulating inflammatory responses as well as cell cycle proliferation and apoptosis. Although TNF stimulates apoptosis, it also activates the transcription factor NF-B, and studies have shown that inhibition of NF-B potentiates the cytotoxicity of TNF. Since several chemotherapy agents act like TNF to both promote apoptosis and activate NF-B, understanding the role of NF-B in suppressing apoptosis may have significant clinical applications. To understand the effects of stimulation with TNF and the role of NF-B in regulating this response, a 23k human cDNA microarray was used to screen TNF-inducible genes in HeLa cells. Real-time PCR verified expression changes in 16 of these genes and revealed three distinct temporal patterns of expression after TNF stimulation. Using RNA interference to disrupt expression of the p65 subunit of NF-B, all but two of the genes were shown to depend on this transcription factor for their expression, which correlated well with the existence of NF-B binding sites in most of their promoters. Inflammatory, proapoptotic, and antiapoptotic genes were all shown to be regulated by NF-B, demonstrating the wide variety of targets activated by NF-B signaling and the necessity of differentiating among these genes for therapeutic purposes.