Novel Insights into the Cellular Mechanisms of the Anti-inflammatory Effects of NF-κB Essential Modulator Binding Domain Peptides

Abstract

The classical nuclear factor κB (NF-κB) signaling pathway is under the control of the IκB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-κB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-κB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of κBα (IκBα). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.

Peptide-based inhibition of IκB kinase/nuclear factor-κB pathway protects against diabetes-associated nephropathy and atherosclerosis in a mouse model of type 1 diabetes

“…These findings are consistent with those of previous studies characterising the in vitro NF-κB blocking effect of different NBD peptide sequences in cytokine-stimulated mononuclear cells, osteoclasts and fibroblasts [26,[35][36][37][38]. Remarkably, NBD peptide reversed the cellular responses induced by long-term exposure to HG, but did not influence either NF-κB or cell viability under normoglycaemic conditions.…”

“…Real-time PCR revealed a dose-dependent inhibition of NF-κB-dependent genes (Ccl2, Ccl5 and Tnfα) by the NBD peptide in HG-stimulated MCT (Fig. 2a, b), with an inhibition efficiency (IC 50 =2.1-3.4 μmol/l) in a similar micromolar range as previously tested [10,26]. In MC, NBD peptide decreased pro-inflammatory gene expression induced by HG and LPS stimulation (Fig.…”

Peptide-Based Inhibition of NF-κB Rescues Diaphragm Muscle Contractile Dysfunction in a Murine Model of Duchenne Muscular Dystrophy

“…We initiated our study by using the same non-GLP version of the NBD peptide that we and many other laboratories have used to demonstrate NF-κB involvement in a specific model of disease (13,23,(32)(33)(34)(35)(36)(37). This peptide is formulated in DMSO and was administered as a 10% solution in saline.…”

“…An IKK complex containing NEMO is critical for inflammatory signaling through the classical NF-κB pathway (13,14). When the association between IKKα and IKKβ and NEMO is disrupted, inflammatory signaling through IKK is impeded.…”

3β-Angeloyloxy-8β,10β-dihydroxyeremophila-7(11)-en-12,8α-lactone Inhibits Lipopolysaccharide-Induced Nitric Oxide Production in RAW264.7 Cells

“…3,14,15,29) Reports exhibited that MAPK pathway positively regulates the stability of several inflammatory mediator mRNAs, including TNF-α and COX-2.…”