ABSTRACT
The activation of nuclear factor kappaB (NFkappaB) is a key event in immune and inflammatory responses. In this study, a cell-penetrating transport peptide, transportan (TP) or its shorter analogue TP 10, was used to facilitate the cellular uptake of an NFkappaB decoy. Peptide nucleic acid (PNA) hexamer or nonamer was linked to the transport peptide by a disulfide bond. NFkappaB decoy oligonucleotide consisted of a double-stranded consensus sequence corresponding to the kappaB site localized in the IL-6 gene promoter, 5'-GGGACTTTCCC-3', with a single-stranded protruding 3'-terminal sequence complementary to the PNA sequence was hybridized to the transport peptide-PNA construct. The ability of the transport peptide-PNA-NFkappaB decoy complex to block the effect of interleukin (IL)-1beta-induced NFkappaB activation and IL-6 gene expression was analyzed by electrophoretic mobility shift assay and reverse transcriptase-polymerase chain reaction in rat Rinm5F insulinoma cells. Preincubation with transport peptide-PNA-NFkappaB decoy (1 microM, 1 h) blocked IL-1beta-induced NFkappaB-binding activity and significantly reduced the IL-6 mRNA expression. The same concentration of NFkappaB decoy in the absence of transport peptide-PNA had no effect even after longer incubations. Our results showed that binding of the oligonucleotide NFkappaB decoy to the nonamer PNA sequence resulted in a stable complex that was efficiently translocated across the plasma membrane.
Subject(s)
Oligodeoxyribonucleotides/genetics , Oligonucleotides/genetics , Peptide Nucleic Acids/genetics , Recombinant Fusion Proteins/genetics , Animals , Base Sequence , Drug Carriers , Electrophoretic Mobility Shift Assay/methods , Galanin , Gene Expression , Interleukin-1/genetics , Interleukin-6/genetics , Models, Genetic , Nucleic Acid Hybridization/genetics , RNA, Messenger/genetics , Rats , Wasp VenomsABSTRACT
betaA25-35, a neurotoxic fragment of the Alzheimer beta-amyloid peptide (betaA), acts as a strong inducer of pro-inflammatory cytokines, such as interleukin (IL)-1 and IL-6, in glial cells. Since IL-1 is known to induce expression of both IL-1 and IL-6, we have investigated to what extent the induction of IL-1alpha and IL-6 by betaA25-35, is dependent on the IL-1 receptor type I (IL-1RI), the only known signalling IL-1 receptor. Primary astroglial cell cultures prepared from wild-type and IL-1RI-deficient mice were incubated in the presence of betaA25-35 (100 microM) for 19 h, followed by analysis of mRNA levels of IL-1alpha and IL-6. Cell cultures treated with betaA25-35 showed a significant increase in mRNA levels for IL-1alpha and IL-6 and in addition increased levels of IL-1alpha immunoreactivity. A supersensitive IL-1alpha response was observed in astroglial cell cultures lacking the IL-1 RI as compared to betaA25-35 treated cell cultures from wild-type mice. In contrast the betaA25-35-induced increase of IL-6 was lower in the absence of IL-1RI. In conclusion, these results suggest that a functional IL-1 signal transduction is not necessary for induction of mRNA levels of IL-1alpha and IL-6 in astroglial cell cultures treated with betaA25-35, but that induction of IL-6 involves at least two distinct mechanisms, one of which occurs via activation of the IL-1RI.
