The IκB-protein BCL-3 controls Toll-like receptor-induced MAPK activity by promoting TPL-2 degradation in the nucleus.

Proinflammatory responses induced by Toll-like receptors (TLRs) are dependent on the activation of the NF-ĸB and mitogen-activated protein kinase (MAPK) pathways, which coordinate the transcription and synthesis of proinflammatory cytokines. We demonstrate that BCL-3, a nuclear IĸB protein that regulates NF-ĸB, also controls TLR-induced MAPK activity by regulating the stability of the TPL-2 kinase. TPL-2 is essential for MAPK activation by TLR ligands, and the rapid proteasomal degradation of active TPL-2 is a critical mechanism limiting TLR-induced MAPK activity. We reveal that TPL-2 is a nucleocytoplasmic shuttling protein and identify the nucleus as the primary site for TPL-2 degradation. BCL-3 interacts with TPL-2 and promotes its degradation by promoting its nuclear localization. As a consequence, Bcl3-/- macrophages have increased TPL-2 stability following TLR stimulation, leading to increased MAPK activity and MAPK-dependent responses. Moreover, BCL-3-mediated regulation of TPL-2 stability sets the MAPK activation threshold and determines the amount of TLR ligand required to initiate the production of inflammatory cytokines. Thus, the nucleus is a key site in the regulation of TLR-induced MAPK activity. BCL-3 links control of the MAPK and NF-ĸB pathways in the nucleus, and BCL-3-mediated TPL-2 regulation impacts on the cellular decision to initiate proinflammatory cytokine production in response to TLR activation.

The Ubiquitination of NF-κB Subunits in the Control of Transcription.

Nuclear factor (NF)-κB has evolved as a latent, inducible family of transcription factors fundamental in the control of the inflammatory response. The transcription of hundreds of genes involved in inflammation and immune homeostasis require NF-κB, necessitating the need for its strict control. The inducible ubiquitination and proteasomal degradation of the cytoplasmic inhibitor of κB (IκB) proteins promotes the nuclear translocation and transcriptional activity of NF-κB. More recently, an additional role for ubiquitination in the regulation of NF-κB activity has been identified. In this case, the ubiquitination and degradation of the NF-κB subunits themselves plays a critical role in the termination of NF-κB activity and the associated transcriptional response. While there is still much to discover, a number of NF-κB ubiquitin ligases and deubiquitinases have now been identified which coordinate to regulate the NF-κB transcriptional response. This review will focus the regulation of NF-κB subunits by ubiquitination, the key regulatory components and their impact on NF-κB directed transcription.

Mapping the Interaction of B Cell Leukemia 3 (BCL-3) and Nuclear Factor κB (NF-κB) p50 Identifies a BCL-3-mimetic Anti-inflammatory Peptide.

The NF-κB transcriptional response is tightly regulated by a number of processes including the phosphorylation, ubiquitination, and subsequent proteasomal degradation of NF-κB subunits. The IκB family protein BCL-3 stabilizes a NF-κB p50 homodimer·DNA complex through inhibition of p50 ubiquitination. This complex inhibits the binding of the transcriptionally active NF-κB subunits p65 and c-Rel on the promoters of NF-κB target genes and functions to suppress inflammatory gene expression. We have previously shown that the direct interaction between p50 and BCL-3 is required for BCL-3-mediated inhibition of pro-inflammatory gene expression. In this study we have used immobilized peptide array technology to define regions of BCl-3 that mediate interaction with p50 homodimers. Our data show that BCL-3 makes extensive contacts with p50 homodimers and in particular with ankyrin repeats (ANK) 1, 6, and 7, and the N-terminal region of Bcl-3. Using these data we have designed a BCL-3 mimetic peptide based on a region of the ANK1 of BCL-3 that interacts with p50 and shares low sequence similarity with other IκB proteins. When fused to a cargo carrying peptide sequence this BCL-3-derived peptide, but not a mutated peptide, inhibited Toll-like receptor-induced cytokine expression in vitro. The BCL-3 mimetic peptide was also effective in preventing inflammation in vivo in the carrageenan-induced paw edema mouse model. This study demonstrates that therapeutic strategies aimed at mimicking the functional activity of BCL-3 may be effective in the treatment of inflammatory disease.

Measurement of NF-κB transcriptional activity and identification of NF-κB cis-regulatory elements using luciferase assays.

The NF-κB family of transcription factors is activated in response to numerous environmental stimuli and coordinates the transcriptional response to immunoreceptors such as the Toll-like receptors, cytokine receptors, and antigen receptors, growth factors, survival factors, and stress signals such as ultraviolet irradiation and oxidative stress. The transcriptional targets of these various pathways include approximately 500 experimentally indentified genes, and it is highly likely that many others remain to be discovered. A genome-wide analysis of NF-κB-chromatin interactions has revealed a surprisingly large number of NF-κB binding sites across the entire genome, many of which are found in intergenic regions and many more do not appear to be associated with changes in transcription of nearby genes. Assessing the consequences of NF-κB binding at genomic sites is therefore essential to determine the functional role of NF-κB in regulating the expression of specific genes. Luciferase-based reporter assays provide a robust and flexible method to test the contribution of specific NF-κB sites to the regulation of gene transcription. The methods described in this chapter may be applied to any promoter sequence and used in a variety of cell lines and conditions to provide critical information on the regulation of gene expression by NF-κB.

Assessing sites of NF-κB DNA binding using chromatin immunoprecipitation.

The NF-κB transcription factor is in fact a family of related proteins which dimerize to form at least 12 distinct complexes which regulate the expression of hundred of genes of importance to a range of physiological and pathological processes. The binding of NF-κB to the regulatory regions and promoters of target genes is influenced by a number of factors including the sequence of DNA-binding sites, the posttranslational modification of NF-κB, and the interaction of cofactors and co-regulators of transcription. In addition, the binding of NF-κB to promoters is highly dynamic and the recruitment of specific subunits to specific binding sites may occur with distinct kinetics. Moreover, genome-wide analysis of NF-κB chromatin binding indicates that the majority of DNA-binding events are not associated with changes in transcriptional activity. Thus, the analysis of NF-κB recruitment and activity at specific binding sites is of critical importance in understanding the regulation of transcription. In this chapter we describe a chromatin immunoprecipitation assay to investigate the in situ binding of NF-κB to specific sites in the genome.

Control of NF-κB subunits by ubiquitination.

NF-κB is an essential regulator of inflammation and is also required for normal immune development and homeostasis. The inducible activation of NF-κB by a wide range of immuno-receptors such as the toll-like receptors (TLR), Tumour Necrosis Factor receptor (TNFR), and antigen T cell and B cell receptors requires the ubiquitin-triggered proteasomal degradation of IκBα to promote the nuclear translocation and transcriptional activity of NF-κB dimers. More recently, an additional role for ubiquitination and proteasomal degradation in the control of NF-κB activity has been uncovered. In this case, it is the ubiquitination and proteasomal degradation of the NF-κB subunits that play a critical role in the termination of the NF-κB-dependent transcriptional response induced by receptor activation. The primary trigger of NF-κB ubiquitination is DNA binding by NF-κB dimers and is further controlled by specific phosphorylation events which regulate the interaction of NF-κB with the E3 ligase complex and the deubiquitinase enzyme USP7. It is the balance between ubiquitination and deubiquitination that shapes the NF-κB-mediated transcriptional response. This chapter describes methods for the analysis of NF-κB ubiquitination.

The Regulation of Endotoxin Tolerance and its Impact on Macrophage Activation.

Endotoxin tolerance in macrophages is a key regulatory mechanism to limit the innate immune response to infection or injury. Long considered a state of unresponsiveness to Toll-like receptor activation, tolerance is now recognized as a state of altered responsiveness to infection or injury. Endotoxin tolerance leads to a shift away from a pro-inflammatory response toward a response with key anti-inflammatory and pro-resolution features. Advances in our understanding of Toll-like receptor function have identified a number of molecular mechanisms that promote tolerance, but how these are integrated to achieve gene-specific regulation is an important outstanding question. The potential to harness the mechanisms of endotoxin tolerance to promote the resolution of chronic inflammation warrants the continued investigation of this fundamental feature of innate immunity. This review focuses on the endotoxin tolerant state, our understanding of the underlying molecular mechanisms, and the clinical significance of endotoxin tolerance.

Inhibition of transcription by B cell Leukemia 3 (Bcl-3) protein requires interaction with nuclear factor κB (NF-κB) p50.

B cell leukemia 3 (Bcl-3) is an essential negative regulator of NF-κB during Toll-like receptor and TNF receptor signaling. Bcl-3 also interacts with a number of transcriptional regulators, including homodimers of the NF-κB p50 subunit. Deletion of Bcl-3 results in increased NF-κB p50 ubiquitination and proteasomal degradation and increased inflammatory gene expression. We employed immobilized peptide array technology to define a region of p50 required for the formation of a Bcl-3·p50 homodimer immunosuppressor complex. Our data demonstrate that amino acids 359-361 and 363 of p50 are critical for interaction with Bcl-3 and essential for Bcl-3-mediated inhibition of inflammatory gene expression. Bcl-3 is unable to interact with p50 when these amino acids are mutated, rendering it incapable of inhibiting the transcriptional activity of NF-κB. Bcl-3 interaction-defective p50 is hyperubiquitinated and has a significantly reduced half-life relative to wild-type p50. Nfkb1(-/-) cells reconstituted with mutated p50 precursor p105 are hyperresponsive to TNFα stimulation relative to wild-type p105, as measured by inflammatory gene expression. Mutant p105 recapitulates a Bcl3(-/-) phenotype. This study demonstrates that interaction with p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3 and further highlights the importance of p50 homodimer stability in the control of NF-κB target gene expression.

Deubiquitination of NF-κB by Ubiquitin-Specific Protease-7 promotes transcription.

NF-κB is the master regulator of the immune response and is responsible for the transcription of hundreds of genes controlling inflammation and immunity. Activation of NF-κB occurs in the cytoplasm through the kinase activity of the IκB kinase complex, which leads to translocation of NF-κB to the nucleus. Once in the nucleus, NF-κB transcriptional activity is regulated by DNA binding-dependent ubiquitin-mediated proteasomal degradation. We have identified the deubiquitinase Ubiquitin Specific Protease-7 (USP7) as a regulator of NF-κB transcriptional activity. USP7 deubiquitination of NF-κB leads to increased transcription. Loss of USP7 activity results in increased ubiquitination of NF-κB, leading to reduced promoter occupancy and reduced expression of target genes in response to Toll-like- and TNF-receptor activation. These findings reveal a unique mechanism controlling NF-κB activity and demonstrate that the deubiquitination of NF-κB by USP7 is critical for target gene transcription.