Transcription Factor Oligodeoxynucleotides to NF-κB Inhibit Transcription of IL-8 in Bronchial Cells

Abstract

Chronic pulmonary inflammation in patients affected by cystic fibrosis (CF) is characterized by massive bronchial infiltrates of neutrophils, which is sustained by the interaction of pathogens (e.g., Pseudomonas aeruginosa) with surface bronchial cells. To explore new treatment options focused on the reduction of neutrophil chemotaxis, we applied the transcription factor (TF) decoy approach, based on the intracellular delivery of double-stranded oligodeoxynucleotides (ODNs) causing inhibition of the binding of TF-related proteins to the different consensus sequences in the promoter of specific genes. In CF bronchial IB3-1 cells, P. aeruginosa induced transcription of the neutrophil chemokines IL-8 and GRO-gamma, of the adhesion molecule intercellular adhesion molecule (ICAM)-1, and of the cytokines IL-1beta and IL-6. Since consensus sequences for the TF, NF-kappaB, are contained in the promoters of all these genes, IB3-1, CuFi-1, Beas-2B, and CaLu-3 cells were transfected with double-stranded TF "decoy" ODNs mimicking different NF-kappaB consensus sequences. IL-8 NF-kappaB decoy ODN partially inhibited the P. aeruginosa-dependent transcription of IL-8, GRO-gamma, and IL-6, whereas decoy ODNs to both HIV-1 long terminal repeat and Igk produced a strong, 80 to 85% inhibition of transcription of IL-8, without reducing that of GRO-gamma, ICAM-1, IL-1beta, and IL-6. In conclusion, intracellular delivery of "decoy" molecules aimed to compete with the TF, NF-kappaB, is a promising strategy to obtain inhibition of IL-8 gene transcription.

Mapping the Transcriptional Machinery of the IL-8 Gene in Human Bronchial Epithelial Cells

“…EMSA experiments were performed as previously described (34). Doublestranded synthetic ODNs, designed on the putative consensus sequences of TFs NF-kB, NF-IL6, AP-1, CHOP, and CREB localized within proximal promoter region of the IL-8 gene (see Table I), were used.…”

“…To address this issue we applied in this study the TF "decoy" strategy (33,34), based on oligodeoxynucleotides (ODNs) mimicking the consensus sequences for the major TFs identified within the proximal promoter region of the IL-8 gene in order to obtain a complete mapping of the TFs involved. In this paper, we report that the induction of IL-8 gene transcription by P. aeruginosa in human bronchial epithelial IB3-1 cells involves a cooperative activation of the MAPKs ERK and p38, as well as their substrate kinases RSK, MSK, and HSP27, and of the TFs NF-kB, NF-IL6, AP-1, CHOP, and CREB.…”

Daedalea gibbosa substances inhibit LPS-induced expression of iNOS by suppression of NF-κB and MAPK activities in RAW 264.7 macrophage cells

“…The electrophoretic mobility shift assay (EMSA) was performed using a doublestranded synthetic oligonucleotides mimicking the NF-κB binding sites (22,23). The synthetic oligonucleotides (Sigma-Genosys, UK) were 5'-end labelled using [Á- 32 P]ATP and T4 poly-nucleotide kinase (MBI Fermentas, Italy).…”

“…The synthetic oligonucleotides (Sigma-Genosys, UK) were 5'-end labelled using [Á- 32 P]ATP and T4 poly-nucleotide kinase (MBI Fermentas, Italy). Binding reactions were set up as described by Bezzerri et al (22) in a total volume of 20 μl containing binding buffer plus 5% glycerol, 1 mM DTT, 0.75 μg of the non-specific competitor poly (dI:dC)·poly(dI:dC), 4 μg of crude nuclear extracts isolated from human K562 cells, increasing amounts of D. gibbosa active fractions (F5-F7), and 0.25 ng of labeled oligonucleotide. In these experiments, the D. gibbosa active fractions were pre-incubated for 20 min with nuclear extracts, before the addition of the labelled target DNA.…”

Transcription factor decoy against NFATc1 in human primary osteoblasts

“…The transcription factor decoy (TFD) strategy is a kind of gene silencing approach aimed at eliminating or attenuating the activity of a specific transcription factor (16)(17)(18)(19)(20)(21)(22)(23). TFD is based on the cellular transfection of double-stranded DNA oligonucleotides (ODNs) mimicking the cis-element that is recognized by the transcription factor, and transcription factor binding on endogenous target genes is quickly and effectively blocked.…”