P50 Function Research Articles

NF-κB1 p50 stabilizes HIF-1α protein through suppression of ATG7-dependent autophagy

The function and underlying mechanisms of p50 in the regulation of protein expression is much less studied because of its lacking of transactivation domain. In this study, we discovered a novel function of p50 in its stabilization of hypoxia-inducible factor 1α (HIF-1α) protein under the condition of cells exposed to arsenic exposure. In p50-deficient (p50−/−) cells, the HIF-1α protein expression was impaired upon arsenic exposure, and such defect could be rescued by reconstitutional expression of p50. Mechanistic study revealed that the inhibition of autophagy-related gene 7 (ATG7)-dependent autophagy was in charge of p50-mediated HIF-1α protein stabilization following arsenic exposure. Moreover, p50 deletion promoted nucleolin (NCL) protein translation to enhance ATG7 mRNA transcription via directly binding transcription factor Sp1 mRNA and increase its stability. We further discovered that p50-mediated miR-494 upregulation gave rise to the inhibition of p50-mediated NCL translation by interacting with its 3’-UTR. These novel findings provide a great insight into the understanding of biomedical significance of p50 protein in arsenite-associated disease development and therapy.

Site-Selective Tyrosine Phosphorylation in the Activation of the p50 Subunit of NF-κB for DNA Binding and Transcription.

The family of NF-κB transcriptional activators controls the expression of many genes, including those involved in cell survival and development. The family consists of homo- and heterodimers constituted by combinations of five subunits. Subunit p50 includes 13 tyrosine residues, but the relationship between specific tyrosine phosphorylations and p50 function is not well understood. Subunits of p50 and p65 prepared in vitro formed a heterodimer, but this NF-κB would not bind to the interleukin-2 (IL-2) promoter DNA. Treatment of p50 with guanosine triphosphate (GTP) and a lysate from activated Jurkat cells, effected rapid p50 phosphorylation, and, in the presence of wild-type subunit p65, was accompanied on the same time scale by IL-2 promoter DNA binding. Modified p50s containing one of seven stoichiometrically phosphorylated tyrosines in NF-κB p50/p65 heterodimers, included three that facilitated binding to the IL-2 DNA promoter region to a greater extent than the wild type. One of these three stoichiometrically phosphorylated p50/p65 heterodimers of NF-κB, containing pTyr60 in the p50 subunit, was treated with a lysate from activated Jurkat cells + GTP and shown to be phosphorylated on the same time scale as wild-type p50. This modified NF-κB also developed IL-2 promoter DNA binding activity on the same time scale as the wild type but exhibited greater binding to the IL-2 DNA promoters than the wild type. The nature of this enhanced binding was studied in greater detail using a metabolically stable pTyr derivative at position 60 of p50 and cellular phosphatases. We suggest that enhanced DNA binding of modified NF-κB containing pTyr60 in the p50 subunit may reflect stoichiometric NF-κB phosphorylation at a site that is not normally fully phosphorylated, or not phosphorylated at all, and is relatively resistant to the effects of Jurkat cell tyrosine phosphatase activity. This conclusion was reinforced by demonstrating that modification of Tyr60 of p50 with a metabolically stable methylenephosphonate moiety further increased the stability of the formed NF-κB p50/p65 heterodimer against the action of activated Jurkat cell phosphatases.

P50 suppresses cytotoxic T lymphocyte effector function to regulate tumor immune escape and response to immunotherapy

BackgroundNF-κB is a key link between inflammation and cancer. Previous studies of NF-κB have largely focused on tumor cells, and the intrinsic function of NF-κB in T cells in tumor...

Loss of NF-\u03baB p50 function synergistically augments microglial priming in the middle-aged brain

BackgroundWhile NF-κB p50 function is impaired in central nervous system disease, aging in non-CNS tissues, and response to reactive oxygen species, the role of NF-κB p50 in aging-associated microglial pro-inflammatory priming is poorly understood.MethodsMale NF-κB p50+/+ and NF-κB p50−/− mice at three different ages (1.5–3.0 month old, 8.0–11.0 month old, and 16.0–18.0 month old) were treated with LPS (5 mg/kg, IP) to trigger peripheral inflammation, where circulating cytokines, neuroinflammation, microglia morphology, and NF-κB p50/p65 function in brain tissue were determined 3 h later.ResultsPeripheral LPS injection in 9-month-old C57BL/6 mice resulted in lower NF-κB p50 DNA binding of nuclear extracts from the whole brain, when compared to 3-week-old C57BL/6 mice, revealing differences in LPS-induced NF-κB p50 activity in the brain across the mouse lifespan. To examine the consequences of loss NF-κB p50 function with aging, NF-κB p50+/+ and NF-κB p50−/− mice of three different age groups (1.5–3.0 month old, 8.0–11.0 month old, and 16.0–18.0 month old) were injected with LPS (5 mg/kg, IP). NF-κB p50−/− mice showed markedly elevated circulating, midbrain, and microglial TNFα when compared to NF-κB p50+/+ mice at all ages. Notably, the 16.0–18.0-month-old (middle aged) NF-κB p50−/− mice exhibited synergistically augmented LPS-induced serum and midbrain TNFα when compared to the younger (1.5–3.0 month old, young adult) NF-κB p50−/− mice. The 16.0–18.0-month-old LPS-treated NF-κB p50−/− mice also had the highest midbrain IL-1β expression, largest number of microglia with changes in morphology, and greatest elevation of pro-inflammatory factors in isolated adult microglia. Interestingly, aging NF-κB p50−/− mice exhibited decreased brain NF-κB p65 expression and activity.ConclusionsThese findings support that loss of NF-κB p50 function and aging in middle-aged mice may interact to excessively augment peripheral/microglial pro-inflammatory responses and point to a novel neuroinflammation signaling mechanism independent the NF-κB p50/p65 transcription factor in this process.

Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus

The genome of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) contains 10 double stranded RNA segments (S1–S10). The segment 7 (S7) encodes 50kDa protein which is considered as a structural protein. The expression pattern and function of p50 in the virus life cycle are still unclear. In this study, the viral structural protein 7 (VP7) polyclonal antibody was prepared with immunized mouse to explore the presence of small VP7 gene-encoded proteins in Bombyx mori cytoplasmic polyhedrosis virus. The expression pattern of vp7 gene was investigated by its overexpression in BmN cells. In addition to VP7, supplementary band was identified with western blotting technique. The virion, BmCPV infected cells and midguts were also examined using western blotting technique. 4, 2 and 5 bands were detected in the corresponding samples, respectively. The replication of BmCPV genome in the cultured cells and midgut of silkworm was decreased by reducing the expression level of vp7 gene using RNA interference. In immunoprecipitation experiments, using a polyclonal antiserum directed against the VP7, one additional shorter band in BmCPV infected midguts was detected, and then the band was analyzed with mass spectrum (MS), the MS results showed thatone candidate interacted protein (VP7 voltage-dependent anion-selective channel-like isoform, VDAC) was identified from silkworm. We concluded that the novel viral product was generated with a leaky scanning mechanism and the VDAC may be an interacted protein with VP7.

NF-κB p50 activation associated with immune dysregulation confers poorer survival for diffuse large B-cell lymphoma patients with wild-type p53

Dysregulated NF-κB signaling is critical for lymphomagenesis, however, the expression and clinical relevance of NF-κB subunit p50 in diffuse large B-cell lymphoma have not been evaluated. In this study, we analyzed the prognostic significance and gene expression signatures of p50 nuclear expression as a surrogate for p50 activation in 465 patients with de novo diffuse large B-cell lymphoma. We found that p50+ nuclear expression, observed in 34.6% of diffuse large B-cell lymphoma, predominantly composed of activated B-cell-like subtype, was an independent adverse prognostic factor in patients with activated B-cell-like diffuse large B-cell lymphoma. It was also an adverse prognostic factor in patients with wild-type TP53 independent of the activated B-cell-like and germinal center B-cell-like subtypes, even though p50 activation correlated with significantly lower levels of Myc, PI3K, phospho-AKT, and CXCR4 expression and less frequent BCL2 translocations. In contrast, in germinal center B-cell-like diffuse large B-cell lymphoma patients with TP53 mutations, p50+ nuclear expression correlated with significantly better clinical outcomes, and decreased p53, Bcl-2, and Myc expression. Gene expression profiling revealed multiple signaling pathways potentially upstream the p50 activation through either canonical or noncanonical NF-κB pathways, and suggested that immune suppression, including that by the immune checkpoint TIM-3 and that through leukocyte immunoglobulin-like receptors, but not antiapoptosis and proliferation, may underlie the observed poorer survival rates associated with p50+ nuclear expression in diffuse large B-cell lymphoma. In conclusion, these data show that p50 is important as a unique mechanism of R-CHOP-resistance in activated B-cell-like diffuse large B-cell lymphoma and in patients without TP53 mutations. The results also provide insights into the regulation and function of p50 in diffuse large B-cell lymphoma and its cross talk with the p53 pathway with important therapeutic implications.

Damaging heterozygous mutations in NFKB1 lead to diverse immunologic phenotypes

The nuclear factor κ light-chain enhancer of activated B cells (NF-κB) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF-κB pathway genes cause immunodeficiency. We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation. We applied genetic linkage analysis and next-generation sequencing and functional analyses of NFKB1 and its mutated alleles. In all affected subjects we detected novel heterozygous variants in NFKB1, encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small-vessel vasculitis characteristic of Behçet disease. Patients with a p.R157X stop-gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome-dependent degradation of both the truncated and wild-type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein-protein interactions. Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behçet disease, can be caused by rare monogenic variants in genes of the NF-κB pathway.

Redox Regulation of the M1/M2 Shift in Microglia: Programming the Deleterious Phenotype

Microglia, the resident innate immune cells in the brain, are implicated in the progressive nature of diverse neurodegenerative diseases associated with aging, including Parkinson's disease. Our recent findings point to the loss of NF‐κB p50 function due to reactive oxygen species as a key mechanism driving microglial failure to resolve the M1 (pro‐inflammatory) response and impaired M2 (alternative) activation, where loss of NF‐κB p50 function leads to a CNS‐specific vulnerability to chronic inflammation. Moreover, our current work indicates that aging synergistically augments the brain M1 activation response in NF‐κB p50−/− mice, implicating a role for loss of NF‐κB p50 function in age‐associated microglial priming (heightened sensitivity to pro‐inflammatory stimuli) and dopaminergic neurotoxicity. Together, these findings implicate NF‐κB p50 as pivotal redox signaling mechanism regulating the M1/M2 activation balance in microglia, where loss of function tips this balance to favor a M1 polarized and neurotoxic phenotype. These findings support that loss of NF‐κB p50 function and aging may interact to excessively augment microglial priming and point to a novel pro‐inflammatory signaling mechanism independent the NF‐κB p50/p65 transcription factor in this process.Support or Funding InformationNIEHS/NIH ONES Award R01ES016951; DOD GW120068

Redox regulation of NF-κB p50 and M1 polarization in microglia.

Redox-signaling is implicated in deleterious microglial activation underlying CNS disease, but how ROS program aberrant microglial function is unknown. Here, the oxidation of NF-κB p50 to a free radical intermediate is identified as a marker of dysfunctional M1 (pro-inflammatory) polarization in microglia. Microglia exposed to steady fluxes of H2 O2 showed altered NF-κB p50 protein-protein interactions, decreased NF-κB p50 DNA binding, and augmented late-stage TNFα expression, indicating that H2 O2 impairs NF-κB p50 function and prolongs amplified M1 activation. NF-κB p50(-/-) mice and cultures exhibited a disrupted M2 (alternative) response and impaired resolution of the M1 response. Persistent neuroinflammation continued 1 week after LPS (1 mg/kg, IP) administration in the NF-κB p50(-/-) mice. However, peripheral inflammation had already resolved in both strains of mice. Treatment with the spin-trap DMPO mildly reduced LPS-induced 22 h TNFα in the brain in NF-κB p50(+/+) mice. Interestingly, DMPO failed to reduce and strongly augmented brain TNFα production in NF-κB p50(-/-) mice, implicating a fundamental role for NF-κB p50 in the regulation of chronic neuroinflammation by free radicals. These data identify NF-κB p50 as a key redox-signaling mechanism regulating the M1/M2 balance in microglia, where loss of function leads to a CNS-specific vulnerability to chronic inflammation.

NF-κB1 inhibits c-Myc protein degradation through suppression of FBW7 expression

NF-κB is a well-known transcription factor in regulation of multiple gene transcription and biological processes, and most of them are relied on its transcriptional activity of the p65/RelA subunit, while biological function of another ubiquitously expressed subunit NF-κB1 (p50) remains largely unknown due to lack transcriptional activation domain. Here we discovered a novel biological function of p50 as a regulator of oncogenic c-Myc protein degradation upon arsenite treatment in a NF-κB transcriptional-independent mechanism. Our results found that p50 was crucial for c-Myc protein induction following arsenite treatment by using specific knockdown and deletion of p50 in its normal expressed cells as well as reconstituting expression of p50 in its deficient cells. Subsequently we showed that p50 upregulated c-Myc protein expression mainly through inhibiting its degradation. We also identified that p50 exhibited this novel property by suppression of FBW7 expression. FBW7 was profoundly upregulated in p50-deficient cells in comparison to that in p50 intact cells, whereas knockdown of FBW7 in p50-/- cells restored arsenite-induced c-Myc protein accumulation, assuring that FBW7 up-regulation was responsible for defect of c-Myc protein expression in p50-/- cells. In addition, we discovered that p50 suppressed fbw7 gene transcription via inhibiting transcription factor E2F1 transactivation. Collectively, our studies demonstrated a novel function of p50 as a regulator of c-Myc protein degradation, contributing to our notion that p50-regulated protein expression through multiple levels at protein translation and degradation, further providing a significant insight into the understanding of biomedical significance of p50 protein.

Tetrahymena telomerase holoenzyme assembly, activation, and inhibition by domains of the p50 central hub.

The eukaryotic reverse transcriptase, telomerase, adds tandem telomeric repeats to chromosome ends to promote genome stability. The fully assembled telomerase holoenzyme contains a ribonucleoprotein (RNP) catalytic core and additional proteins that modulate the ability of the RNP catalytic core to elongate telomeres. Electron microscopy (EM) structures of Tetrahymena telomerase holoenzyme revealed a central location of the relatively uncharacterized p50 subunit. Here we have investigated the biochemical and structural basis for p50 function. We have shown that the p50-bound RNP catalytic core has a relatively low rate of tandem repeat synthesis but high processivity of repeat addition, indicative of high stability of enzyme-product interaction. The rate of tandem repeat synthesis is enhanced by p50-dependent recruitment of the holoenzyme single-stranded DNA binding subunit, Teb1. An N-terminal p50 domain is sufficient to stimulate tandem repeat synthesis and bridge the RNP catalytic core, Teb1, and the p75 subunit of the holoenzyme subcomplex p75/p19/p45. In cells, the N-terminal p50 domain assembles a complete holoenzyme that is functional for telomere maintenance, albeit at shortened telomere lengths. Also, in EM structures of holoenzymes, only the N-terminal domain of p50 is visible. Our findings provide new insights about subunit and domain interactions and functions within the Tetrahymena telomerase holoenzyme.

NFκB1 (p50) suppresses SOD2 expression by inhibiting FoxO3a transactivation in a miR190/PHLPP1/Akt-dependent axis

The biological functions of nuclear factor κB1 (NFκB1; p50) have not been studied as often as those of other members of the NFκB family due to its lack of a transcriptional domain. Our recent studies showed that p50 functions as an apoptotic mediator via its inhibition of GADD45α protein degradation and increase in p53 protein translation. Here we report a novel function of p50 in its regulation of superoxide dismutase 2 (SOD2) transcription via an NFκB-independent pathway. We find that deletion of p50 in mouse embryonic fibroblasts (MEFs; p50(-/-)) up-regulates SOD2 expression at both protein and mRNA levels. SOD2 promoter-driven luciferase is also up-regulated in p50(-/-) cells compared with wild-type (WT) MEF (p50(+/+)) cells, suggesting p50 regulation of SOD2 at the transcriptional level. Our results also show that p50 deficiency specifically results in down-regulation of phosphorylation and increased transactivation of FoxO3a compared with WT cells. Further studies indicate that p50-down-regulated FoxO3a phosphorylation is mediated by activated Akt via up-regulation of microRNA 190 (miR190), in turn inhibiting PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) translation. Together our studies identify a novel p50 function in the regulation of SOD2 transcription by modulating the miR190/PHLPP1/Akt-FoxO3a pathway, which provides significant insight into the physiological function of p50.

NF-κB1 p50 promotes p53 protein translation through miR-190 downregulation of PHLPP1

The biological function of NF-κB1 (p50) in the regulation of protein expression is far from well understood owing to the lack of a transcriptional domain. Here, we report a novel function of p50 in its regulation of p53 protein translation under stress conditions. We found that the deletion of p50 (p50-/-) impaired arsenite-induced p53 protein expression, which could be restored after reconstitutive expression of HA-p50 in p50-/- cells, p50-/-(Ad-HA-p50). Further studies indicated that the amounts of p53 mRNA, p53 promoter-driven transcription activity and p53 protein degradation were comparable between wild-type and p50-/- cells. Moreover, we found that p50 was crucial for Akt/S6 ribosomal protein activation via inhibition of the translation of the PH domain and leucine-rich repeat protein phosphatases 1 (PHLPP1), a phosphatase of Akt. Further studies showed that p50-mediated upregulation of miR-190 was responsible for the inhibition of PHLPP1 translation by targeting the 3'-untranslated region of its mRNA. Collectively, we have identified a novel function of p50 in modulating p53 protein translation via regulation of the miR-190/PHLPP1/Akt-S6 ribosomal protein pathway.

The NF-κB p50 Subunit Is Protective during Intestinal Entamoeba histolytica Infection of 129 and C57BL/6 Mice

Entamoeba histolytica is the agent of amebic colitis. In this work we examined the intestinal NF-kappaB response to this parasite. Using an enzyme-linked immunosorbent assay (ELISA) and an electrophoretic mobility shift assay, we found that the NF-kappaB subunit p50 predominated in nuclear extracts of whole cecal tissue and of isolated crypts from mice inoculated with E. histolytica. p50 was protective, since C57BL/6 and 129 mice in which there was targeted deletion of this subunit were more susceptible to E. histolytica infection as measured by culture results, cecal parasite ELISA results, and/or histologic scores. The transepithelial electrical resistance of cecal explants from C57BL/6 and 129 p50 knockout mice decreased markedly in response to the parasite compared with the transepithelial electrical resistance of their wild-type counterparts, suggesting that a protective function of p50 was present in the epithelium itself. This work shows that NF-kappaB activity, particularly activity of the p50 subunit, is one factor that contributes to resistance of the gut to E. histolytica infection.

Interaction of the universal mRNA-binding protein, p50, with actin: a possible link between mRNA and microfilaments.

We have shown previously that p50 is the most abundant protein associated with a variety of eukaryotic mRNAs and exhibits about 98% amino acid sequence identity to mammalian Y-box binding transcription factors. The dual function of p50 in the cell as a regulator of both transcription and translation has been suggested. To gain insight into the role of p50 in these processes, we performed the yeast two-hybrid screen to identify p50 molecular partners. Here we report the identification of actin as a p50-interacting protein. Coimmunoprecipitation of p50 and actin from HeLa extracts as well as in vitro binding studies indicate specificity and a high affinity for the interaction between p50 and actin. Interestingly, p50 binding to actin is affected by mRNA; binding was observed at a low p50/mRNA ratio and was greatly reduced at higher ratios. Since the p50/mRNA ratio appears to be important for mRNA translatability, we speculate that p50 can regulate the attachment of mRNA to the actin network depending on its translational activity. Using immunofluorescence, we show that p50 binds to actin filaments in permeabilized cells and causes actin fibers to bundle in vitro. Together, these findings support the view that p50 may play an important role in mRNA transport, anchoring, and localization on actin filaments in the cell.