Stepwise phosphorylation of p65 promotes NF-kappa B activation and NK cell responses during target cell recognition

NF-κB is a key transcription factor that dictates the outcome of diverse immune responses. How NF-κB is regulated by multiple activating receptors that are engaged during natural killer (NK)-target cell contact remains undefined. Here we show that sole engagement of NKG2D, 2B4 or DNAM-1 is insufficient for NF-κB activation. Rather, cooperation between these receptors is required at the level of Vav1 for synergistic NF-κB activation. Vav1-dependent synergistic signalling requires a separate PI3K-Akt signal, primarily mediated by NKG2D or DNAM-1, for optimal p65 phosphorylation and NF-κB activation. Vav1 controls downstream p65 phosphorylation and NF-κB activation. Synergistic signalling is defective in X-linked lymphoproliferative disease (XLP1) NK cells entailing 2B4 dysfunction and required for p65 phosphorylation by PI3K-Akt signal, suggesting stepwise signalling checkpoint for NF-κB activation. Thus, our study provides a framework explaining how signals from different activating receptors are coordinated to determine specificity and magnitude of NF-κB activation and NK cell responses

Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65.

Human bocavirus (HBoV), a parvovirus, is a single-stranded DNA etiologic agent causing lower respiratory tract infections in young children worldwide. Nuclear factor kappa B (NF-κB) transcription factors play crucial roles in clearance of invading viruses through activation of many physiological processes. Previous investigation showed that HBoV infection could significantly upregulate the level of TNF-α which is a strong NF-κB stimulator. Here we investigated whether HBoV proteins modulate TNF-α-mediated activation of the NF-κB signaling pathway. We showed that HBoV NS1 and NS1-70 proteins blocked NF-κB activation in response to TNF-α. Overexpression of TNF receptor-associated factor 2 (TRAF2)-, IκB kinase alpha (IKKα)-, IκB kinase beta (IKKβ)-, constitutively active mutant of IKKβ (IKKβ SS/EE)-, or p65-induced NF-κB activation was inhibited by NS1 and NS1-70. Furthermore, NS1 and NS1-70 didn't interfere with TNF-α-mediated IκBα phosphorylation and degradation, nor p65 nuclear translocation. Coimmunoprecipitation assays confirmed the interaction of both NS1 and NS1-70 with p65. Of note, NS1 but not NS1-70 inhibited TNF-α-mediated p65 phosphorylation at ser536. Our findings together indicate that HBoV NS1 and NS1-70 inhibit NF-κB activation. This is the first time that HBoV has been shown to inhibit NF-κB activation, revealing a potential immune-evasion mechanism that is likely important for HBoV pathogenesis

Pericyte NF-κB Activation Enhances Endothelial Cell Proliferation and Proangiogenic Cytokine Secretion in Vitro

Pericytes are skeletal muscle resident, multipotent stem cells that are localized to the microvasculature. In vivo, studies have shown that they respond to damage through activation of nuclear-factor kappa-B (NF-κB), but the downstream effects of NF-κB activation on endothelial cell proliferation and cell–cell signaling during repair remain unknown. The purpose of this study was to examine pericyte NF-κB activation in a model of skeletal muscle damage; and use genetic manipulation to study the effects of changes in pericyte NF-κB activation on endothelial cell proliferation and cytokine secretion. We utilized scratch injury to C2C12 cells in coculture with human primary pericytes to assess NF-κB activation and monocyte chemoattractant protein-1 (MCP-1) secretion from pericytes and C2C12 cells. We also cocultured endothelial cells with pericytes that expressed genetically altered NF-κB activation levels, and then quantified endothelial cell proliferation and screened the conditioned media for secreted cytokines. Pericytes trended toward greater NF-κB activation in injured compared to control cocultures (P = 0.085) and in comparison to C2C12 cells (P = 0.079). Second, increased NF-κB activation in pericytes enhanced the proliferation of cocultured endothelial cells (1.3-fold,P = 0.002). Finally, we identified inflammatory signaling molecules, including MCP-1 and interleukin 8 (IL-8) that may mediate the crosstalk between pericytes and endothelial cells. The results of this study show that pericyte NF-κB activation may be an important mechanism in skeletal muscle repair with implications for the development of therapies for musculoskeletal and vascular diseases, including peripheral artery disease

Interferon-γ Activates Nuclear Factor-κ B in Oligodendrocytes through a Process Mediated by the Unfolded Protein Response

Our previous studies have demonstrated that the effects of the immune cytokine interferon-γ (IFN-γ) in immune-mediated demyelinating diseases are mediated, at least in part, by the unfolded protein response (UPR) in oligodendrocytes. Data indicate that some biological effects of IFN-γ are elicited through activation of the transcription factor nuclear factor-κB (NF-κB). Interestingly, it has been shown that activation of the pancreatic endoplasmic reticulum kinase (PERK) branch of the UPR triggers NF-κB activation. In this study, we showed that IFN-γ-induced NF-κB activation was associated with activation of PERK signaling in the oligodendroglial cell line Oli-neu. We further demonstrated that blockage of PERK signaling diminished IFN-γ-induced NF-κB activation in Oli-neu cells. Importantly, we showed that NF-κB activation in oligodendrocytes correlated with activation of PERK signaling in transgenic mice that ectopically express IFN-γ in the central nervous system (CNS), and that enhancing IFN-γ-induced activation of PERK signaling further increased NF-κB activation in oligodendrocytes. Additionally, we showed that suppression of the NF-κB pathway rendered Oli-neu cells susceptible to the cytotoxicity of IFN-γ, reactive oxygen species, and reactive nitrogen species. Our results indicate that the UPR is involved in IFN-γ-induced NF-κB activation in oligodendrocytes and suggest that NF-κB activation by IFN-γ represents one mechanism by which IFN-γ exerts its effects on oligodendrocytes in immune-mediated demyelinating diseases

Optineurin Is Required for CYLD-Dependent Inhibition of TNFα-Induced NF-κB Activation

The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP

Mouse models for aberrant NF-kB activation in B-cell development and lymphomagenesis

The NF-κB family of transcription factors promotes the expression of survival, proliferation, inflammation, and differentiation programs in B-cells as well as in other cells of the immune system. Additionally, its aberrant constitutive activation is a hallmark of several B-cell neoplasms. Moreover, recurrent genetic lesions targeting the NF-κB activation pathways have been identified in patient samples from different B-cell neoplasms. For instance, genetic abnormalities in A20, the negative regulator of the canonical NF-κB pathway, and the alternative NF-κB arm TRAF3/NIK have been reported in splenic marginal zone lymphoma (sMZL). In contrast, few genetic lesions affecting NF-κB activation have been detected in chronic lymphocytic leukaemia (CLL), albeit the observed constitutive NF-κB activation in CLL. It has been proposed that signals from the microenvironment might promote NF-κB activation in CLL. Taken together, the recurrent observation of enhanced or constitutive NF-κB activation and the broad spectrum of genetic lesions targeting NF-κB in B-cell neoplasm, strongly suggest that NF-κB activation could act as a general mechanism in B-cell transformation. To date, there is little evidence linking activation of the NF-κB pathways directly to B-cell transformation and there are few available in vivo models addressing the role of NF-κB activation in B-cell lymphomagenesis that bring a better understanding of the disease development, progression and therapeutic strategies. Therefore, the objective of this thesis was to investigate the role of aberrant NF-κB activation in B-cell transformation and lymphomagenesis using available mouse models. First, the potential cooperation between canonical and alternative NF-κB activation in predisposing mice to sMZL was investigated by making use of the published mouse strains mimicking the chromosomal gains in NIK and deletions in A20 observed in human sMZL patients. Second, the direct effect of constitutive canonical NF-κB activation in B-cell transformation was investigated by conditionally expressing an IKK2 constitutive active mutant (IKK2ca) in B-cells, and in the Eμ-TCL1tg mouse model for human CLL. As expected, the hemizygous ablation of A20 cooperated with NIK overexpression in expanding the marginal zone B-cell (MZB) pool in mice. However, homozygous ablation of A20 in combination with NIK overexpression resulted in an unexpected impaired mature B-cell homeostasis that was evident by a systemic depletion of mature B-cells in secondary lymphoid organs and recirculating B-cells in the bone marrow. Moreover, loss of A20 aggravated the previously reported block in adaptive immunity imposed by the overexpression of NIK in B-cells. Aberrant NF-κB activation by ablation of A20 and NIK overexpression resulted in an abnormal pre-activated antigen presenting cell phenotype in B-cells that was accompanied by the altered expression of integrins, important for retaining cells in the MZ. Furthermore, the impairment in B-cell homeostasis was accompanied by the expansion of regulatory CD25+ CD4 T-cells in addition to effector-like CD4 and CD8 T-cells. This T-cell hyperplasia was maintained in aged mice that developed an expansion of myeloid cells with age. The concomitant effector T-cell hyperplasia and later expansion of myeloid cells, suggest a possible involvement of these cells in the observed reduced B-cell numbers. It seems that aberrant canonical and alternative NF-κB activation in B-cells affect the terminal differentiation of mature B-cells forcing cells into a stage that fails to terminally differentiate into follicular B-cells and MZB-cells. Moreover, their aberrant phenotype possibly triggered a T-cell dependent immune response that may be involved in the elimination of these aberrant B-cells. On the other hand, constitutive canonical NF-κB activation by expression of the IKK2ca mutant in B-cells resulted in the expansion of B1a-cells that with age developed into a disease reminiscent of human small lymphocytic lymphoma - a form of human CLL - where CD5+ B-cells infiltrated and accumulated in different lymphoid compartments. Moreover, constitutive NF-κB activation in B-cells shortened mice life span in an IKK2ca dose dependent manner. Furthermore, expression of IKK2ca in B-cells cooperated with the TCL1tg oncogene in accelerating the disease progression in Eμ-TCL1tg CLL mouse model also in a dose dependent manner. Strikingly, similar survival dynamics were observed when the conditional IKK2ca allele was activated during early B-cell development (CD19cre) or in a small percentage of mature B-cells (Cγ1cre or AIDcre). Finally, adoptive transplant experiments demonstrated that constitutive NF-κB activation failed to compensate for microenvironment dependent-signals required for the proper support of CLL-cells in vivo. In conclusion, aberrant NF-κB activation in B-cells had different effects that were dependent on the mechanism of NF-κB activation and transcriptional programs this regulated. While ablation of A20 in combination with NIK overexpression in B-cells impaired mature B-cell homeostasis; IKK2ca-dependent constitutive NF-κB activation promoted the development of B-cell neoplasms in mice and collaborated with the TCL1 oncogene accelerating CLL progression in mice

Imaging Pulmonary NF-kappaB Activation and Therapeutic Effects of MLN120B and TDZD-8

NF-κB activation is a critical signaling event in the inflammatory response and has been implicated in a number of pathological lung diseases. To enable the assessment of NF-κB activity in the lungs, we transfected a luciferase based NF-κB reporter into the lungs of mice or into Raw264.7 cells in culture. The transfected mice showed specific luciferase expression in the pulmonary tissues. Using these mouse models, we studied the kinetics of NF-κB activation following exposure to lipopolysaccharide (LPS). The Raw264.7 cells expressed a dose-dependent increase in luciferase following exposure to LPS and the NF-κB reporter mice expressed luciferase in the lungs following LPS challenge, establishing that bioluminescence imaging provides adequate sensitivity for tracking the NF-κB activation pathway. Interventions affecting the NF-κB pathway are promising clinical therapeutics, thus we further examined the effect of IKK-2 inhibition by MLN120B and glycogen synthase kinase 3 beta inhibition by TDZD-8 on NF-κB activation. Pre-treatment with either MLN120B or TDZD-8 attenuated NF-κB activation in the pulmonary tissues, which was accompanied with suppression of pro-inflammatory chemokine MIP-1ß and induction of anti-inflammatory cytokine IL-10. In summary, we have established an imaging based approach for non-invasive and longitudinal assessment of NF-κB activation and regulation during acute lung injury. This approach will potentiate further studies on NF-κB regulation under various inflammatory conditions

Diverse roles of the ubiquitin system in NF-κB activation.

NF-κB is a transcription factor known to be involved in pleomorphic biological phenomena such as inflammation and immune responses. Abnormal activation of NF-κB has been reported in many pathological conditions, including allergic and auto-inflammatory diseases and malignancies. Therefore, the NF-κB activation pathway has been extensively studied and involvement of the ubiquitin conjugation system in the NF-κB activation pathways has been shown. Also non-degradable roles of the ubiquitin system have been revealed, recently. Several types of polyubiquitin chains exist and the type of chain seems to determine how ubiquitinated proteins are regulated. Roles of non-degradable types of polyubiquitin chains such as K63, linear and K11 chains in NF-κB activation is one of the big issues in NF-κB research. Thus, this short article discusses the differential roles of those polyubiquitin chains in NF-κB activation. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf