Inhibition of NF-κB by MG132 through ER stress-mediated induction of LAP and LIP

Proteasome inhibitor MG132 blocks activation of NF-κB by preventing degradation of IκB. In this report, we propose an alternative mechanism by which MG132 inhibits cytokine-triggered NF-κB activation. We found that MG132 induced endoplasmic reticulum (ER) stress, and attenuation of ER stress blunted the suppressive effect of MG132 on NF-κB. Through ER stress, MG132 up-regulated C/EBPβ mRNA transiently and caused sustained accumulation of its translational products liver activating protein (LAP) and liver-enriched inhibitory protein (LIP), both of which were identified as suppressors of NF-κB. Our results disclosed a novel mechanism underlying inhibition of NF-κB by MG132.     

Loss of desmin triggers mechanosensitivity and up-regulation of Ankrd1 expression through Akt-NF-κB signaling pathway in smooth muscle cells

Muscle cells, including human airway smooth muscle cells (HASMCs) express ankyrin repeat protein 1 (Ankrd1), a member of ankyrin repeat protein family. Ankrd1 efficiently interacts with the type III intermediate filament desmin. Our earlier study showed that desmin is an intracellular load-bearing protein that influences airway compliance, lung recoil, and airway contractile responsiveness. These results suggest that Ankrd1 and desmin may play important roles on ASMC homeostasis. Here we show that small interfering (si)RNA-mediated knockdown of the desmin gene in HASMCs, recombinant HASMCs (reHASMCs), up-regulates Ankrd1 expression. Moreover, loss of desmin in HASMCs increases the phosphorylation of Akt, inhibitor of κB kinase (IKK)-α, and inhibitor of κB (IκB)-α proteins, leading to NF-κB activation. Treatment of reHASMCs with Akt, IKKα, IκBα, or NF-κB inhibitor inhibits the loss of desmin-induced Ankrd1 up-regulation, suggesting Akt/NF-κB-mediated Ankrd1 regulation. Transfection of reHASMCs with siRNA specific for p50 or p65 corroborates the NF-κB-mediated Ankrd1 regulation. Luciferase reporter assays show that NF-κB directly binds on Ankrd1 promoter and up-regulates Ankrd1 levels. Overall, our data provide a new link between desmin and Ankrd1 regulation, which may be important for ASMC homeostasis.     

Possible participation of intracellular platelet-activating factor in NF-κB activation in rat peritoneal macrophages

As we had found previously that thapsigargin, an endomembrane Ca²⁺-ATPase inhibitor, induces production of intracellular platelet-activating factor (PAF) [Br. J. Pharmacol. 116 (1995) 2141], we decided to investigate the possible roles of intracellular PAF in nuclear factor (NF)-κB activation of thapsigargin-stimulated rat peritoneal macrophages. When rat peritoneal macrophages were stimulated with thapsigargin, the level of inhibitory protein of NF-κB-α (IκB-α) was decreased and the nuclear translocation of NF-κB was increased. The thapsigargin-induced activation of NF-κB was inhibited by the PAF synthesis inhibitor SK&F 98625 and the PAF antagonist E6123. Structurally unrelated PAF antagonists such as E5880 and L-652,731 also inhibited the thapsigargin-induced activation of NF-κB. Lipopolysaccharide (LPS)-induced activation of NF-κB was also suppressed by these drugs. In a culture of rat peritoneal macrophages, exogenously added PAF did not induce degradation of IκB-α. These findings suggest that the intracellular PAF produced by the stimulation with thapsigargin or LPS is involved in activation of the NF-κB pathway.     

Non-canonical NF-κB signaling pathway

The non-canonical NF-κB pathway is an important arm of NF-κB signaling that predominantly targets activation of the p52/RelB NF-κB complex. This pathway depends on the inducible processing of p100, a molecule functioning as both the precursor of p52 and a RelB-specific inhibitor. A central signaling component of the non-canonical pathway is NF-κB-inducing kinase (NIK), which integrates signals from a subset of TNF receptor family members and activates a downstream kinase, IκB kinase-α (IKKα), for triggering p100 phosphorylation and processing. A unique mechanism of NIK regulation is through its fate control: the basal level of NIK is kept low by a TRAF-cIAP destruction complex and signal-induced non-canonical NF-κB signaling involves NIK stabilization. Tight control of the fate of NIK is important, since deregulated NIK accumulation is associated with lymphoid malignancies.     

Suppression of NF-κB signaling by KEAP1 regulation of IKKβ activity through autophagic degradation and inhibition of phosphorylation

IκB kinase β (IKKβ) plays a crucial role in biological processes, including immune response, stress response, and tumor development by mediating the activation of various signaling molecules such as NF-κB. Extensive studies on the mechanisms underlying IKK activation have led to the identification of new activators and have facilitated an understanding of the cellular responses related to NF-κB and other target molecules. However, the molecular processes that modulate IKK activity are still unknown. In this study, we show that KEAP1 is a new IKK binding partner, which is responsible for the down-regulation of TNFα-stimulated NF-κB activation. The E(T/S)GE motif, which is found only in the IKKβ subunit of the IKK complex, is essential for interaction with the C-terminal Kelch domain of KEAP1. Reduction of KEAP1 expression by small interfering RNA enhanced NF-κB activity, and up-regulated the expression of NF-κB target genes. Ectopic expression of KEAP1 decreased the expression of IKKβ, which was restored by an autophagy inhibitor. IKK phosphorylation stimulated by TNFα was blocked by KEAP1. Our data demonstrate that KEAP1 is involved in the negative regulation of NF-κB signaling through the inhibition of IKKβ phosphorylation and the mediation of autophagy-dependent IKKβ degradation.