Inhibition of TNF-α gene expression and bioactivity by site-specific transcription factor-binding oligonucleotides

Abstract

The present study investigated transcriptional inactivation of TNF-α gene by nuclear factor-binding oligonucleotides (ON) and their effects on pulmonary inflammatory responses in mice. PCR-based gene mutation and gel shift assays were used to identify specific cis-acting elements necessary for nuclear factor binding and transactivation of TNF-α gene by lipopolysaccharide (LPS). LPS inducibility of TNF-α was shown to require transcriptional activation by NF-κB at multiple binding sites, including the −850 (κ1), −655 (κ2), and −510 (κ3) sites, whereas the −210 (κ4) site had no effect. Maximum inducibility was associated with the activation of κ3 site. The sequence-specific, double-stranded ON targeting this site was most effective in inhibiting TNF-α activity induced by LPS. The inhibitory effect of ON on TNF-α bioactivity was also investigated using a murine lung inflammation model. Pretreatment of mice with ON, but not its mutated sequence, inhibited LPS-induced inflammatory neutrophil influx and TNF-α production by lung cells. Effective inhibition by ON in this model was shown to require a liposomal agent for efficient cellular delivery of the ON. Together, our results indicate that transcriptional inactivation of TNF-α gene can be achieved by using ON that compete for nuclear factor binding to TNF-α gene promoter. This gene inhibition approach may be used as a research tool or as potential therapeutic modality for diseases with etiology dependent on aberrant gene expression.

p85α Acts as a Novel Signal Transducer for Mediation of Cellular Apoptotic Response to UV Radiation

“…MEFs were stably transfected with the TNF-␣-luciferase reporter plasmid, in which the transcription of the luciferase reporter gene was driven by the mouse TNF-␣ promoter (nucleotides Ϫ1260 to ϩ60) (56,59). We found that the TNF-␣ promoter-driven luciferase activities were significantly induced in wild-type MEFs in a dose-dependent manner but that such induction was dramatically suppressed in p85␣ Ϫ/Ϫ transfectants under the same experimental conditions (Fig.…”

“…The TNF-␣-luciferase reporter plasmid, in which the transcription of the luciferase reporter gene is driven by the upstream 5Ј-flanking region of the TNF-␣ gene promoter (nucleotides Ϫ1260 to ϩ60), has been described previously (56,59). The NF-B-, AP-1-, and NFATluciferase reporter plasmids were also described previously (23,55).…”

Role of TNF-α in vascular dysfunction

“…The production of ROS can stimulate a cytokine cascade through NF-κB-induced transcriptional events, which then induce the expression of TNF-α [31,32]. TNF-α stimulates O 2 •− production in neutrophils and ECs, reportedly via CAPK (ceramide-activated protein kinase), NADPH oxidase [33], XO [34], NOS [35,36] etc.…”

Transcriptional Control of the TNF Gene

“…Although mutations in these low-affinity NF-κB sites were reported to reduce LPS inducibility of the human TNF gene by 40–50% in reporter assays [29, 87], these reports were hard to reconcile with studies showing that mutation of κ1 [37] and even deletion of the entire distal promoter [22, 37–39, 41] did not impact LPS induction of a human TNF reporter gene. Similarly, in gene reporter assays with the murine TNF promoter, deletion of the distal promoter region [42] and mutation of κB1 (and in some cases, κB3, κB2, and κB2a) had little to no effect on LPS-induced TNF transcription [29, 99]. Thus, there has been no clear functional role demonstrated for these NF-κB motifs in the activation of murine or human TNF gene transcription.…”