The IκB kinase complex in NF-κB regulation and beyond

The I{kappa}B kinase (IKK) complex is the signal integration hub for NF-{kappa}B activation. Composed of two serine-threonine kinases (IKK{alpha} and IKK{beta}) and the regulatory subunit NEMO (also known as IKK{gamma}), the IKK complex integrates signals from all NF-{kappa}B activating stimuli to catalyze the phosphorylation of various I{kappa}B and NF-{kappa}B proteins, as well as of other substrates. Since the discovery of the IKK complex components about 15 years ago, tremendous progress has been made in the understanding of the IKK architecture and its integration into signaling networks. In addition to the control of NF-{kappa}B, IKK subunits mediate the crosstalk with other pathways, thereby extending the complexity of their biological function. This review summarizes recent advances in IKK biology and focuses on emerging aspects of IKK structure, regulation and function

Deficiency in IκBα in the intestinal epithelium leads to spontaneous inflammation and mediates apoptosis in the gut

The IκB-Kinase (IKK)-NF-κB signalling pathway plays a multifaceted role in inflammatory bowel disease (IBD): on the one hand, it protects from apoptosis; on the other, it activates transcription of numerous inflammatory cytokines and chemokines. Although several murine models of IBD rely on disruption of IKK-NF-κB signalling, these involve either knockouts of a single family-member of NF-κB, or of upstream kinases that are known to have additional, NF-κB-independent, functions. This has made the distinct contribution of NF-κB to homeostasis in intestinal epithelium cells difficult to assess. To examine the role of constitutive NF-κB activation in intestinal epithelial cells, we generated a mouse model with a tissue-specific knockout of the direct inhibitor of NF-κB, Nfkbia/IκBα. We demonstrate that constitutive activation of NF-κB in intestinal epithelial cells induces several hallmarks of IBD including increased apoptosis, mucosal inflammation in both the small intestine and the colon, crypt hyperplasia, and depletion of Paneth cells, concomitant with aberrant Wnt signalling. To determine which NF-κB-driven phenotypes are cell-intrinsic, and which are extrinsic and thus require the immune compartment, we established a long-term organoid culture. Constitutive NF-κB promoted stem-cell proliferation, mis-localisation of Paneth cells, and sensitisation of intestinal epithelial cells to apoptosis in a cell-intrinsic manner. Increased number of stem cells was accompanied by a net increase in Wnt activity in organoids. Because aberrant Wnt signalling is associated with increased risk of cancer in IBD patients and because NFKBIA has recently emerged as a risk locus for IBD, our findings have critical implications for the clinic. In a context of constitutive NF-κB, our findings imply that general anti-inflammatory or immunosuppressive therapies should be supplemented with direct targeting of NF-κB within the epithelial compartment in order to attenuate apoptosis, inflammation, and hyperproliferation

A quantitative modular modeling approach reveals the effects of different A20 feedback implementations for the NF-κB signaling dynamics

Signaling pathways involve complex molecular interactions and are controled by non-linear regulatory mechanisms. If details of regulatory mechanisms are not fully elucidated, they can be implemented by different, equally reasonable mathematical representations in computational models. The study presented here focusses on NF-κB signaling, which is regulated by negative feedbacks via IκBα and A20. A20 inhibits NF-κB activation indirectly through interference with proteins that transduce the signal from the TNF receptor complex to activate the IκB kinase (IKK) complex. A number of pathway models has been developed implementing the A20 effect in different ways. We here focus on the question how different A20 feedback implementations impact the dynamics of NF-κB. To this end, we develop a modular modeling approach that allows combining previously published A20 modules with a common pathway core module. The resulting models are fitted to a published comprehensive experimental data set and therefore show quantitatively comparable NF-κB dynamics. Based on defined measures for the initial and long-term behavior we analyze the effects of a wide range of changes in the A20 feedback strength, the IκBα feedback strength and the TNFα stimulation strength on NF-κB dynamics. This shows similarities between the models but also model-specific differences. In particular, the A20 feedback strength and the TNFα stimulation strength affect initial and long-term NF-κB concentrations differently in the analyzed models. We validated our model predictions experimentally by varying TNFα concentrations applied to HeLa cells. These time course data indicate that only one of the A20 feedback models appropriately describes the impact of A20 on the NF-κB dynamics in this cell type

TSG101 associates with PARP1 and is essential for PARylation and DNA damage-induced NF-κB activation

In a genome-wide screening for components of the dsDNA-break-induced IKK-NF-κB pathway, we identified scores of regulators, including tumor susceptibility gene TSG101. TSG101 is essential for DNA damage-induced formation of cellular poly(ADP-ribose) (PAR). TSG101 binds to PARP1 and is required for PARP1 activation. This function of TSG101 is independent of its role in the ESCRT-I endosomal sorting complex. In the absence of TSG101, the PAR-dependent formation of a nuclear PARP1-IKKγ signalosome, which triggers IKK activation, is impaired. According to its requirement for PARP1 and NF-κB activation, TSG101-deficient cells are defective in DNA repair and apoptosis protection. Loss of TSG101 results in PARP1 trapping at damage sites and mimics the effect of pharmacological PARP inhibition. We also show that the loss of TSG101 in connection with inactivated tumor suppressors BRCA1/2 in breast cancer cells is lethal. Our results imply TSG101 as a therapeutic target to achieve synthetic lethality in cancer treatment

RC3H1 post-transcriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-kappa B pathway

The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNF alpha mRNA decay via a 3'UTR constitutive decay element (CDE). Here we applied PAR-CLIP to human RC3H1 to identify similar to 3, 800 mRNA targets with 416, 000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage-induced mRNAs, indicating a role of this RNA-binding protein in the post-transcriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of the NF-kappa B pathway regulators such as I kappa B alpha and A20. RC3H1 uses ROQ and Zn-finger domains to contact a binding site in the A20 30UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with I kappa B kinase and NF-kappa B activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-kappa B pathway

NEMO oligomerization and its ubiquitin-binding properties

The IKK [IκB (inhibitory κB) kinase] complex is a key regulatory component of NF-κB (nuclear factor κB) activation and is responsible for mediating the degradation of IκB, thereby allowing nuclear translocation of NF-κB and transcription of target genes. NEMO (NF-κB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKα and IKKβ, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKβ disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKα and IKKβ and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO–di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-κB signalling

High expression of RelA/p65 is associated with activation of nuclear factor-κB-dependent signaling in pancreatic cancer and marks a patient population with poor prognosis

Activation of nuclear factor-κB (NF-κB) signaling was observed in pancreatic adenocarcinoma cell lines and tumours. However, information on the expression of RelA/p65, the major transcription activating NF-κB subunit, in these carcinomas and possible correlations thereof with NF-κB activation and patient survival is not available. To provide this missing translational link, we analysed expression of RelA/p65 in 82 pancreatic adenocarcinomas by immunohistochemistry. Moreover, we measured activation of the NF-κB pathway in 11 tumours by quantitative PCR for NF-κB target genes. We observed strong cytoplasmic or nuclear expression of RelA/p65 in 42 and 37 carcinomas, respectively. High cytoplasmic and nuclear expression of RelA/p65 had negative prognostic impact with 2-year survival rates for patients without cytoplasmic or nuclear RelA/p65 positivity of 41 and 40% and rates for patients with strong cytoplasmic or nuclear RelA/p65 expression of 22 and 20%, respectively. High RelA/p65 expression was correlated to increased expression of NF-κB target genes. The observation that high expression of RelA/p65 is correlated to an activation of the NF-κB pathway and indicates poor patient survival identifies a patient subgroup that might particularly benefit from NF-κB-inhibiting agents in the treatment of pancreatic cancer. Based on our findings, this subgroup could be identified by applying simple immunohistochemical techniques