Multiple NF-κB Enhancer Elements Regulate Cytokine Induction of the Human Inducible Nitric Oxide Synthase Gene

Abstract

The human inducible nitric oxide synthase (iNOS) gene is overexpressed in a number of human inflammatory diseases. Previously, we observed that the human iNOS gene is transcriptionally regulated by cytokines and demonstrated that the cytokine-responsive regions are upstream of ؊3.8 kilobase pairs (kb). Therefore, the purpose of this study was to further localize the functional enhancer elements and to assess the role of the transcription factor NF-B in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-B inhibitors significantly suppressed cytokinestimulated iNOS mRNA expression and NO synthesis, indicating that NF-B is involved in the induction of the human iNOS gene. Analysis of the first 4.7 kb of the 5-flanking region demonstrated basal promoter activity and failed to show any cytokine-inducible activity. However, promoter constructs extending to ؊5.8 and ؊7.2 kb revealed 2-3-fold and 4 -5-fold induction, respectively, in the presence of cytokines. DNA sequence analysis from ؊3.8 to ؊7.2 kb identified five putative NF-B cis-regulatory transcription factor binding sites upstream of ؊4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-B motif at ؊5.8 kb is required for cytokine-induced promoter activity, while the sites at ؊5.2, ؊5.5, and ؊6.1 kb elicit a cooperative effect. Electromobility shift assays using a site-specific oligonucleotide and nuclear extracts from cells stimulated with cytokine-mixture, tumor necrosis factor-␣ or interleukin-1␤, but not interferon-␥, exhibited inducible DNA binding activity for NF-B. These data indicate that NF-B activation is required for cytokine induction of the human iNOS gene and identifies four NF-B enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-␣ and interleukin-1␤.

Glucagon-like peptide-1 prevents beta cell glucolipotoxicity

“…Upon its activation, NF-κB will translocate to the nucleus to promote expression of stress-inducible genes that can be either pro-or anti-apoptotic, depending on cell system and stimuli [62,63,64,65]. For instance, NF-κB binding sites have been found in the promoter regions of pro-apoptotic genes like inducible nitric oxide synthase [66], cytokines [67], fas and fas-ligand [68]. Examples of anti-apoptotic proteins whose expression is increased by NF-κB are: caspase inhibitors IAP-1, IAP-2 and XIAP [49], mitochondria membrane stabilisers such as Bcl-2 [50], Bcl-xl and Bfl-1 [69], and cell-cycle regulatory proteins like cyclin D1 [70,71].…”

Role of pro-oxidants and antioxidants in the anti-inflammatory and apoptotic effects of curcumin (diferuloylmethane)

“…NF-κB is known to regulate the expression of proinflammatory and proliferative markers, including iNOS [36], COX-2 [37], MMP-9 [38], and cyclin D1 [39]. To determine whether GSH can inhibit the curcumin to suppress these gene products, cells were pretreated with various concentrations of GSH for 2 h, treated with curcumin for 4 h, and then exposed to TNF.…”

Transfection of NFκB-decoy oligodeoxynucleotides using efficient ultrasound-mediated gene transfer into donor kidneys prolonged survival of rat renal allografts

“…1,39 Transcriptional activation of the genes encoding these adhesion molecules and cytokines is tightly regulated by a transcription factor, NFkB. 4,6,[9][10][11]13,40,41 Under normal conditions, NFkB is usually present with IkB in the cytosol as an inactive complex. However, when grafts are exposed to stimuli after transplantation such as ischemia/reperfusion injury, cells may be activated to promote IkB-phosphorylation, which induces NFkB to translocate to the nucleus and to bind to the promoter region of genes of several major inflammatory mediators, such as IL-1, iNOS, and TNF-a, and upregulate their transcription.…”

“…However, when grafts are exposed to stimuli after transplantation such as ischemia/reperfusion injury, cells may be activated to promote IkB-phosphorylation, which induces NFkB to translocate to the nucleus and to bind to the promoter region of genes of several major inflammatory mediators, such as IL-1, iNOS, and TNF-a, and upregulate their transcription. [5][6][7][8][9][10][11][12][13][14] These products may also promote NFkB activation in a paracrine or autocrine fashion, and this in turn further upregulates the production of chemokines and the expression of surface molecules, establishing the so-called 'cytokine-adhesion molecule cascade', which further augments such inflammatory reaction and promotes the development of acute rejection. [41][42][43][44][45] As we previously reported, NFkB-decoy, which effectively binds NFkB and inhibits its activity, blocks the transactivation of genes for essential cytokines and adhesion molecules in vivo in several animal models of inflammatory disease as well as in vitro.…”

“…2,3 Nuclear factor kB (NFkB) is one of such transcription factors for genes whose products are critical for inflammation and immunity. 4 It rapidly translocates from the cytoplasm to the nucleus in response to a variety of extracellular signals and plays a pivotal role in the regulation of inflammatory changes by inducing coordinated transactivation of genes of major inflammatory mediators such as IL-1, IL-6, iNOS, and TNF-a [5][6][7][8][9][10][11] and adhesion molecules such as ICAM-1 and VCAM-1. [12][13][14] Cooper et al demonstrated a time-dependent increase in the DNA-binding activity of NFkB, which peaked at 3 days before rejection, in a rat cardiac allograft model.…”