期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 280, 期 17, 页码 17435-17448出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M500437200
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资金
- NIAID NIH HHS [R01 AI40218] Funding Source: Medline
- NIEHS NIH HHS [P30 ES06676] Funding Source: Medline
Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappa B (NF-kappa B) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappa B-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappa B inhibitor to systematically identify NF-kappa B-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappa B 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-kappa B-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappa B-dependent genes and five genes not previously known to be NF-kappa B-dependent (Gro-beta and-gamma, I kappa B epsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappa B regulated. Expression of constitutively active enhanced green fluorescent center dot NF-kappa B/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-kappa B sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous I kappa B alpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappa B/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappa B-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.
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