Inhibition of TNF-alpha gene expression and bioactivity by site-specific transcription factor-binding oligonucleotides.
The present study investigated transcriptional inactivation of TNF-alpha 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-alpha gene by lipopolysaccharide (LPS). LPS inducibility of TNF-alpha was shown to require transcriptional activation by NF-kappaB at multiple binding sites, including the -850 (kappa1), -655 (kappa2), and -510 (kappa3) sites, whereas the -210 (kappa4) site had no effect. Maximum inducibility was associated with the activation of kappa3 site. The sequence-specific, double-stranded ON targeting this site was most effective in inhibiting TNF-alpha activity induced by LPS. The inhibitory effect of ON on TNF-alpha 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-alpha 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-alpha gene can be achieved by using ON that compete for nuclear factor binding to TNF-alpha 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.
Ye
Jianping
J
Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA 70808, USA.
Wang
Liying
L
Zhang
Xiaoying
X
Tantishaiyakul
Vimon
V
Rojanasakul
Yon
Y
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism* / Q000494:pharmacology*; D011014:Pneumonia / Q000139:chemically induced / Q000517:prevention & control; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism*
D008070:Lipopolysaccharides; D016328:NF-kappa B; D009841:Oligonucleotides; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha