Journal
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
Volume 284, Issue 2, Pages L386-L394Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajplung.00134.2002
Keywords
tumor necrosis factor-alpha
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Funding
- NHLBI NIH HHS [HL 62959] Funding Source: Medline
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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.
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