The IKKβ Subunit of IκB Kinase (IKK) is Essential for Nuclear Factor κB Activation and Prevention of Apoptosis

The IκB kinase (IKK) complex is composed of three subunits, IKKα, IKKβ, and IKKγ (NEMO). While IKKα and IKKβ are highly similar catalytic subunits, both capable of IκB phosphorylation in vitro, IKKγ is a regulatory subunit. Previous biochemical and genetic analyses have indicated that despite their similar structures and in vitro kinase activities, IKKα and IKKβ have distinct functions. Surprisingly, disruption of the Ikkα locus did not abolish activation of IKK by proinflammatory stimuli and resulted in only a small decrease in nuclear factor (NF)-κB activation. Now we describe the pathophysiological consequence of disruption of the Ikkβ locus. IKKβ-deficient mice die at mid-gestation from uncontrolled liver apoptosis, a phenotype that is remarkably similar to that of mice deficient in both the RelA (p65) and NF-κB1 (p50/p105) subunits of NF-κB. Accordingly, IKKβ-deficient cells are defective in activation of IKK and NF-κB in response to either tumor necrosis factor α or interleukin 1. Thus IKKβ, but not IKKα, plays the major role in IKK activation and induction of NF-κB activity. In the absence of IKKβ, IKKα is unresponsive to IKK activators

Evolution of the POU1F1 transcription factor in mammals: rapid change of the alternatively-spliced β-domain

The POU1F1 (Pit-1) transcription factor is important in regulating expression of growth hormone, prolactin and TSH β-subunit, and controlling development of the anterior pituitary cells in which these hormones are produced. POU1F1 is a conserved protein comprising three main domains, an N-terminal transcription activation domain (TAD), a POU-specific domain and a C-terminal homeodomain. Within the TAD, a β-domain can be inserted by alternative splicing, giving an extended 'β-variant' with altered properties. Here sequence data from over 100 species were used to assess the variability of POU1F1 in mammals. This showed that the POU-specific domain and homeodomain are very strongly conserved, and that the TAD is somewhat less conserved, as are linker and hinge regions between these main domains. On the other hand, the β-domain is very variable, apparently evolving at a rate not significantly different from that expected for unconstrained, neutral evolution. In several species stop and/or frameshift mutations within the β domain would prevent expression of the β-variant as a functional protein. In most species expression of the β-variant is low (<5% of total POU1F1 expression). The rate of evolution of POU1F1 in mammals shows little variation, though the lineage leading to dog does show an episode of accelerated change. This comparative genomics study suggests that in most mammalian species POU1F1 variants produced by alternative splicing may have little physiological significance

IKKβ Activation Is Sufficient for RANK-Independent Osteoclast Differentiation and Osteolysis

Monocytes differentiate into osteoclasts through stimulation of receptor activator of NF-κB (RANK). Many downstream effectors of RANK play a positive role in osteoclastogenesis, but their relative importance in osteoclast differentiation is unclear. We report the discovery that activation of a single pathway downstream of RANK is sufficient for osteoclast differentiation. In this regard, introduction of constitutively activated IKKβ (IKKβSSEE) but not wild-type IKKβ into monocytes stimulates differentiation of bona fide osteoclasts in the absence of RANK ligand (RANKL). This phenomenon is independent of upstream signals because IKKβSSEE induced the development of bone-resorbing osteoclasts from RANK and IKKα knockout monocytes and in conditions in which NEMO-IKKβ association was inhibited. NF-κB p100 and p105, but not RelB, were critical mediators of this effect. Inflammatory autocrine signaling by tumor necrosis factor α (TNF-α) and interleukin 1 (IL-1) were dispensable for the spontaneous osteoclastogenesis driven by IKKβSSEE. More important, adenoviral gene transfer of IKKβSSEE induced osteoclasts and osteolysis in calvariae and knees of mice. Our data establish the sufficiency of IKKβ activation for osteolysis and suggest that IKKβ hyperactivation may play a role in conditions of pathologic bone destruction refractory to RANK/RANKL proximal therapeutic interventions. © 2010 American Society for Bone and Mineral Research

Constitutive intestinal NF-κB does not trigger destructive inflammation unless accompanied by MAPK activation

Constitutive NF-κB activation in IECs induces inflammatory cytokines and chemokines in the lamina propria, but does not result in overt tissue damage unless acute inflammatory insults are present, causing TNF-dependent destruction and barrier disruption

Nitric Oxide-Induced Activation of the AMP-Activated Protein Kinase α2 Subunit Attenuates IκB Kinase Activity and Inflammatory Responses in Endothelial Cells

BACKGROUND: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFκB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). METHODOLOGY/PRINCIPAL FINDINGS: Overexpression of a dominant negative AMPKα2 mutant in tumor necrosis factor-α-stimulated human endothelial cells resulted in increased NFκB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKα2(-/-) mice the interleukin (IL)-1β induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFκB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKα2(-/-) mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IκB and p65, indicating a link between AMPK and the IκB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKα2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKα2(+/+) versus AMPKα2(-/-) mice. CONCLUSIONS: These results demonstrate that the IKK is a direct substrate of AMPKα2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFκB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK

Mechanism of PP2A-mediated IKKβ dephosphorylation: a systems biological approach

BACKGROUND: Biological effects of nuclear factor-kappaB (NF kappaB) can differ tremendously depending on the cellular context. For example, NF kappaB induced by interleukin-1 (IL-1) is converted from an inhibitor of death receptor induced apoptosis into a promoter of ultraviolet-B radiation (UVB)-induced apoptosis. This conversion requires prolonged NF kappaB activation and is facilitated by IL-1 + UVB-induced abrogation of the negative feedback loop for NF kappaB, involving a lack of inhibitor of kappaB (I kappaB alpha) protein reappearance. Permanent activation of the upstream kinase IKK beta results from UVB-induced inhibition of the catalytic subunit of Ser-Thr phosphatase PP2A (PP2Ac), leading to immediate phosphorylation and degradation of newly synthesized I kappaB alpha. RESULTS: To investigate the mechanism underlying the general PP2A-mediated tuning of IKK beta phosphorylation upon IL-1 stimulation, we have developed a strictly reduced mathematical model based on ordinary differential equations which includes the essential processes concerning the IL-1 receptor, IKK beta and PP2A. Combining experimental and modelling approaches we demonstrate that constitutively active, but not post-stimulation activated PP2A, tunes out IKK beta phosphorylation thus allowing for I kappaB alpha resynthesis in response to IL-1. Identifiability analysis and determination of confidence intervals reveal that the model allows reliable predictions regarding the dynamics of PP2A deactivation and IKK beta phosphorylation. Additionally, scenario analysis is used to scrutinize several hypotheses regarding the mode of UVB-induced PP2Ac inhibition. The model suggests that down regulation of PP2Ac activity, which results in prevention of I kappaB alpha reappearance, is not a direct UVB action but requires instrumentality. CONCLUSION: The model developed here can be used as a reliable building block of larger NF kappa B models and offers comprehensive simplification potential for future modeling of NF kappa B signaling. It gives more insight into the newly discovered mechanisms for IKK deactivation and allows for substantiated predictions and investigation of different hypotheses. The evidence of constitutive activity of PP2Ac at the IKK complex provides new insights into the feedback regulation of NF kappa B, which is crucial for the development of new anti-cancer strategies

Deregulated expression of TANK in glioblastomas triggers pro-tumorigenic ERK1/2 and AKT signaling pathways

Signal transmission by the noncanonical IkappaB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IKKɛ, requires interaction with adapter proteins such as TRAF associated NF-κB activator (TANK). Although increased expression or dysregulation of both kinases has been described for a variety of human cancers, this study shows that deregulated expression of the TANK protein is frequently occurring in glioblastomas (GBMs). The functional relevance of TANK was analyzed in a panel of GBM-derived cell lines and revealed that knockdown of TANK arrests cells in the S-phase and prohibits tumor cell migration. Deregulated TANK expression affects several signaling pathways controlling cell proliferation and the inflammatory response. Interference with stoichiometrically assembled signaling complexes by overexpression or silencing of TANK prevented constitutive interferon-regulatory factor 3 (IRF3) phosphorylation. Knockdown of TANK frequently prevents constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). TANK-mediated ERK1/2 activation is independent from the canonical MAP kinase or ERK kinase (MEK) 1/2-mediated pathway and utilizes an alternative pathway that uses a TBK1/IKKɛ/Akt signaling axis, thus identifying a novel pathway suitable to block constitutive ERK1/2 activity.Peer reviewe

Brn2 Is a Transcription Factor Regulating Keratinocyte Differentiation with a Possible Role in the Pathogenesis of Lichen Planus

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. In this study, we investigated the role of the POU domain-containing transcription factor Brn2 in keratinocyte differentiation. Immunohistochemical analysis showed that Brn2 is expressed primarily in the upper granular layer. Consistent with its epidermal localization, Brn2 expression was highly induced at 14 days after calcium treatment of cultured normal human epidermal keratinocytes. When Brn2 was overexpressed by adenoviral transduction, Brn2 led to increased expression of the differentiation-related genes involucrin, filaggrin, and loricrin in addition to inhibition of their proliferation. Chromatin immunoprecipitation demonstrated that Brn2 bound to the promoter regions of these differentiation-related genes. We injected the purified Brn2 adenovirus into rat skin, which led to a thickened epidermis with increased amounts of differentiation related markers. The histopathologic features of adenovirus-Brn2 injected skin tissues looked similar to the features of lichen planus, a human skin disease showing chronic inflammation and well-differentiated epidermal changes. Moreover, Brn2 is shown to be expressed in almost all cell nuclei of the thickened epidermis of lichen planus, and Brn2 also attracts T lymphocytes. Our results demonstrate that Brn2 is probably a transcriptional factor playing an important role in keratinocyte differentiation and probably also in the pathogenesis of lichen planus lesions

Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1β instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation