NF-kappaB Decoy Oligonucleotides Research Articles

Up-Regulation of Histamine H4Receptors Contributes to Splenic Apoptosis in Septic Mice: Counteraction of the Antiapoptotic Action of Nuclear Factor-κB

The histamine H(4) receptor is the most recently identified receptor and is considered to play a role in a variety of inflammatory diseases. Histamine levels in the plasma are known to be elevated in animal models of sepsis and in septic patients. The aim of this study was to test the hypothesis that the H(4) receptor may play a significant role in the pathophysiology of sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture in BALB/c mice. Although the H(4) receptor gene was undetectable in normal peripheral key organs, with the exception of the spleen, the expression levels of this gene were highly up-regulated in all those organs of septic mice. In vivo transfection of nuclear factor-kappaB (NF-kappaB) decoy oligodeoxynucleotide, but not of its scrambled form, resulted in a great inhibition of sepsis-induced overexpression of the H(4) receptor gene. In septic mice, marked increases in caspase-3 activation and follicular lymphocyte apoptosis in spleens were strongly suppressed by systemic treatment with synthetic small interfering RNA (siRNA) targeted to the H(4) receptor. This was associated with the up-regulation of a number of antiapoptotic proteins. These antiapoptotic effects of H(4) receptor siRNA treatment were all inhibited by further application of NF-kappaB decoy oligonucleotide. Our results suggest that superinduction of the histamine H(4) receptor gene in peripheral key organs, including the spleen, that is promoted by sepsis is transcriptionally controlled by NF-kappaB, whereas stimulation of this receptor is involved in the development of sepsis-induced splenic apoptosis through counteraction of the antiapoptotic action of NF-kappaB.

NF-κB Decoy Oligonucleotides Suppress RANTES Expression and Monocyte Chemotactic Activity via NF-κB Inactivation in Stromal Cells of Ectopic Endometrium

The nuclear factor kappa B (NF-kappaB) pathway is a critical mediator of regulated on activation, normal T cell expressed and secreted (RANTES) gene regulation and therefore represents a potential target for therapy of endometriosis-associated symptoms. The objective of this study was to investigate the effect of NF-kappaB decoy oligonucleotides (ODNs) on NF-kappaB activation, RANTES expression, and monocyte chemotactic activity in ectopic endometrial stromal cells in vitro. A specific sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantify RANTES expression in ectopic and normal endometrial stromal cells stimulated by interleukin (IL)-1beta. Four hours after transfection of NF-kappaB decoy ODNs, 10 ng/ml IL-1beta was added to induce the ectopic endometrial stromal cells to secrete RANTES. The NF-kappaB activation, RANTES expression, and monocyte chemotactic activity in ectopic endometrial stromal cells were respectively evaluated by electrophoretic mobility shift assay, ELISA, and Boyden chambers. IL-1beta induced significantly higher levels (P < 0.05) of RANTES expression in a time-dependent manner in ectopic endometrial stromal cells compared with IL-1beta-untreated ectopic and normal endometrial stromal cells. The RANTES accounts for the majority (68%) of the monocyte chemotactic activity in conditioned media of ectopic endometrial stromal cells. In vitro transfection of NF-kappaB decoy ODNs dramatically decreased (P < 0.05) the NF-kappaB activation, RANTES expression, and monocyte chemotactic activity in IL-1beta-induced ectopic endometrial stromal cells. NF-kappaB decoy ODNs may exert anti-inflammatory effects in ectopic endometrial stromal cells via the suppression of NF-kappaB activation, RANTES expression, and monocyte chemotactic activity.

Enhancement of ultraviolet-induced apoptosis by NF-kappaB decoy oligonucleotides

A decoy strategy utilizing oligonucleotides (ODN) containing the specific binding sequence of a certain transcription factor has been developed and is considered to be a potential new class of antigene therapy. However, the application of this new therapeutic modality to skin diseases has not been fully documented. The aim of this work was to examine the effects of the nuclear factor (NF)-kappaB decoy ODN on UV-elicited skin change. Mouse keratinocyte Pam 212 cells were transfected with NF-kappaB decoy ODN to examine the effects of the decoy ODN on ultraviolet (UV) B-induced apoptosis. Tape-stripped rat dorsal skin was treated with an ointment containing NF-kappaB decoy ODN for the examination of the in vivo impact of the decoy ODN on sunburned cell (SBC) formation and UVB erythema. NF-kappaB decoy ODN specifically induced apoptosis of Pam 212 cells and SBC formation was significantly enhanced by topical NF-kappaB decoy ODN ointment, while UV-induced erythema was not affected. These data suggest that enhancement of UV-induced apoptosis by NF-kappaB decoy ODN may play a cancer-preventive role by further eliminating photodamaged keratinocytes.

Sequestration of serum response factor in the hippocampus impairs long‐term spatial memory

The formation of long-term memory has been shown to require protein kinase-mediated gene expression. One such kinase, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), can lead to the phosphorylation of serum response factor (SRF) and Elk-1, enhancing the expression of target genes. However, a direct involvement of these transcription factors in memory storage has not been demonstrated. We have employed an oligonucleotide decoy technique to interrogate SRF and Elk-1. Previously, it has been shown that intra-amygdalal infusion of small double-stranded decoy oligonucleotides for nuclear factor-kappaB (NFkappaB) can impair long-term memory for fear-potentiated startle. Using this approach, we found that intra-hippocampal infusion of NFkappaB decoy oligonucleotides also impairs long-term spatial memory, consistent with a role for this factor in long-term memory storage. Decoy oligonucleotides containing the binding site for SRF, as confirmed by shift-western, did not influence memory acquisition but impaired long-term spatial memory. Analysis of search behavior during the transfer test revealed deficits consistent with a loss of precise platform location information. In contrast, oligonucleotides with a binding site for either Elk-1 or another target of ERK activity, SMAD3/SMAD4, did not interfere with memory formation or storage. These findings suggest that SRF-mediated gene expression is required for long-term spatial memory.

Cellular delivery of a double-stranded oligonucleotide

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.

Retroinfusion of NFkappaB decoy oligonucleotide extends cardioprotection achieved by CD18 inhibition in a preclinical study of myocardial ischemia and retroinfusion in pigs.

Myocardial reperfusion injury is partially mediated by postischemic inflammation. Beyond acute PMN recruitment, postischemic inflammation comprises subacute PMN adhesion, eg via NFkappaB activation. In a pig model of 60-min LAD occlusion by PTCA ballon inflation and 1 to 7 days of reperfusion, we investigated the impact of targeted NFkappaB decoy oligonucleotide (ODN) transfection in the area at risk (AAR) on infarct size and regional myocardial function. After 55 min of LAD occlusion, liposomes containing NFkappaB ODN were selectively retroinfused into the anterior interventricular vein for 5 min. Then, retroinfusion was stopped and reperfusion was initiated. Where indicated, CD18 antibody IB4 was infused systemically at 30 min of ischemia. Methylen blue and tetrazolium-red staining were used for quantification of the infarct size. Subendocardial segment shortening (SES) by sonomicrometric crystals in infarct area and AAR was assessed under pacing (expressed as % of control region). NFkappaB decoy ODN retroinfusion reduced infarct size (36 +/- 4% versus 49 +/- 5% in control hearts at day 7), whereas functional reserve of the AAR (SES 73 +/- 17% versus 46 +/- 18% at 180/min) tended to improve. Similar effects were observed after IB4 infusion (38 +/- 5% infarct size, 85 +/- 7% SES at 180/min). A combination of NFkappaB decoy ODN retroinfusion and IB4 infusion further decreased infarct size (26 +/- 2%) and improved functional reserve (SES 94 +/- 6% at 180/min). We conclude that NFkappaB decoy ODN transfection by retroinfusion is feasible in pig hearts and provides postischemic cardioprotection in addition to CD18 blockade.

Evidence that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-κB

The transcription factor NF-kappaB is a positive transcription factor for a number of genes and has been recognized as an anti-apoptotic regulator. However, the mechanism by which NF-kappaB blocks apoptosis is still controversial. Here, we demonstrate the evidence that NF-kappaB could attenuate the TNF-alpha-induced apoptosis without de novo protein synthesis using human pancreatic cancer cell lines, MIA PaCa-2 and Capan-2. The TNF-alpha-induced apoptosis was blocked by IL-1beta, a potent inducer of NF-kappaB activation. This inhibitory effect of IL-1beta was evident when cells were treated with protein synthesis inhibitors such as cycloheximide (CHX). Moreover, NF-kappaB decoy oligonucleotides could not block the anti-apoptotic effect of IL-1beta at doses sufficient to block the NF-kappaB-dependent transcription induced by IL-1beta. To confirm the role of NF-kappaB in blocking apoptosis, we generated stable cell lines expressing IkappaBdeltaN, a highly stable form of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB. In these stable transfectants, the antiapoptotic effect of IL-1beta was totally abolished, indicating that the anti-apoptotic action of IL-1beta could be ascribed to the NF-kappaB action. These findings show that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-kappaB and support the possibility that NF-kappaB could exert its anti-apoptotic action through protein-protein interaction.