Identification of Direct Genomic Targets Downstream of the Nuclear Factor-κB Transcription Factor Mediating Tumor Necrosis Factor Signaling

Abstract

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-B (NF-B) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-B-dependent genes is unknown. In this study, we used a tetracyclineregulated cell line expressing an NF-B inhibitor to systematically identify NF-B-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-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-B-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-B-dependent genes and five genes not previously known to be NF-B-dependent (Gro-␤ and-␥, IB⑀, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-B regulated. Expression of constitutively active enhanced green fluorescent⅐NF-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-B sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IB␣/⑀, Gro-␤/␥, and Naf-1 promoters directly bound NF-B/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-B-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

ABINs: A20 binding inhibitors of NF-κB and apoptosis signaling

“…Indeed, an NF-B responsive element has been identified in the human ABIN-1 gene promoter and chromatin immunoprecipitation analysis showed binding of p65 to this NF-B site in TNF-treated HeLa cells. Furthermore, overexpression of p65 led to an increase of ABIN-1 mRNA expression in these cells [17]. Similarly, retroviral overexpression of p50 and p65 upregulates ABIN-1 mRNA in primary human keratinocytes, whereas ABIN-1 expression in fibroblasts is unaffected, indicative for a cell type specific NF-B dependent inducibility of ABIN-1 [18].…”

Dual role of TMS1/ASC in death receptor signaling

“…Fas ligand is upregulated by NF-kB and AP-1 following T-cell activation, and the subsequent Fas-mediated cell death is dependent upon intact NF-kB signaling (Kasibhatla et al, 1998(Kasibhatla et al, , 1999. Indeed, recent profiling studies examining the spectrum of TNFa-induced, NF-kB-regulated genes include several with pro-apoptotic functions (Zhou et al, 2003;Tian et al, 2005). TMS1 may be another example of an NF-kB/JNK-regulated factor that functions in a proapoptotic capacity.…”

Galectin-1 Couples Glycobiology to Inflammation in Osteoarthritis through the Activation of an NF-κB–Regulated Gene Network

“…This signature highly overlaps with expression profiles in inflammation. Various types of cells and tissues react this way when stimulated with TNF-a, IL-1, LPS, and IKK inhibitors (47,48), pointing to Galectin-1 as a bona fide proinflammatory coordinator in human chondrocytes. Moreover, the fact that the top six TFBS most significantly associated with genes induced by Galectin-1 are binding sites for NF-kB further intimates the notion that NF-kB is a mediator of the Galectin-1-triggered proinflammatory activities.…”