Abstract
Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F)<0.001) and demonstrated a change in signal intensity of+/-3-fold relative to control. Of these, 28 were NF-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.
Highlights
Tumor necrosis factor (TNF)1 is a pleiotropic cytokine that mediates inflammatory signaling in infectious diseases [1]
Upon its ligation to cell surface receptors, TNF induces protein recruitment to cytoplasmic death domains, assembling a signaling complex composed of TRADD, FADD, TNF receptor-associated factor-2 (TRAF2) that activate divergent intracellular signals, including the c-Jun NH2-terminal kinase-activating protein-1 (AP-1) and the IKK-nuclear factor-B (NF-B) pathways to induce genomic responses in the target cell [8, 10]
The IKK-NF-B pathway is critical for inducing tissue inflammation and preventing TNF-induced programmed cell death, surprisingly little is known about its downstream gene targets
Summary
Cell Culture, Treatment, and Transfection—The human cervical epithelioid carcinoma cell line HeLa expressing tTA (HeLa Tet-Off) pBIEGFP1⁄7IB␣ Mut was constructed earlier [23]. Applied Biosystems assays-by-design or assays-on-demand 20Ј assay mix of primers and TaqMan MGB probes (FAMTM dye-labeled) for all our target genes and predeveloped 18 S rRNA (VICTM-dye labeled probe) TaqManTM assay reagent (P/N 4319413E) for internal control were used for real-time PCR measurements. These assays were designed to span exon-exon junctions so as not to detect genomic DNA.
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