RESUMEN
NF-κB (Nuclear factor-kappa B) family proteins are versatile transcription factors that play crucial regulatory roles in cell development, growth, apoptosis, inflammation, and immune response. However, there is limited research on the function of these key genes in echinoderms. In this study, an NF-κB family gene (SiRel) was identified in sea urchin Strongylocentrotus intermedius. The gene has an open reading frame length of 1809 bp and encodes for 602 amino acids. Domain prediction results revealed that the N-terminal of SiRel protein encodes a conserved Rel homology domain (RHD), including the RHD-DNA binding domain and the RHD-dimerization domain. Multiple sequence comparison results showed that the protein sequences of these two domains were conserved. Phylogenetic analysis indicated that SiRel clustered with Strongylocentrotus purpuratus p65 protein and Rel protein of other echinoderms. Results from quantitative real-time PCR demonstrated detectable SiRel mRNA expression in all tested sea urchin tissues, with the highest expression level found in the gills. And SiRel mRNA expression levels were significantly induced after LPS (Lipopolysaccharide) and poly(I:C) (Polyinosinic:polycytidylic acid) stimulation. In addition, SiRel protein expression can be found in cytoplasm and nucleus of HEK293T cells. Co-immunoprecipitation results showed that SiRel could interact with sea urchin IκB (Inhibitor of NF-κB) protein. Western blotting and dual-luciferase reporter gene assay results indicated that overexpression of SiRel in HEK293T cells could impact the phosphorylation levels of JNK (c-Jun N-terminal kinase) and Erk1/2 (Extracellular signal-regulated kinases1/2) and activate interleukin-6 (IL-6), activating protein 1 (AP-1), interferon (IFN)α/ß/γ, and signal transducer and activator of transcription 3 (STAT3) reporter genes in HEK293T cells. In conclusion, this study reveals that SiRel plays an important role in the innate immune response of sea urchins and enriches our understanding of comparative immunology theory.
Asunto(s)
Secuencia de Aminoácidos , Regulación de la Expresión Génica , Inmunidad Innata , Lipopolisacáridos , Filogenia , Poli I-C , Alineación de Secuencia , Strongylocentrotus , Animales , Inmunidad Innata/genética , Poli I-C/farmacología , Lipopolisacáridos/farmacología , Strongylocentrotus/genética , Strongylocentrotus/inmunología , Alineación de Secuencia/veterinaria , Regulación de la Expresión Génica/inmunología , Clonación Molecular , Perfilación de la Expresión Génica/veterinaria , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , FN-kappa B/inmunología , Secuencia de Bases , Proteínas Proto-Oncogénicas c-rel/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Células HEK293RESUMEN
Antibody responses require the proliferative expansion of B cells controlled by affinity-dependent signals. Yet, proliferative bursts are heterogeneous, varying between 0 and 8 divisions in response to the same stimulus. NFκB cRel is activated in response to immune stimulation in B cells and is genetically required for proliferation. Here, we asked whether proliferative heterogeneity is controlled by natural variations in cRel abundance. We developed a fluorescent reporter mTFP1-cRel for the direct observation of cRel in live proliferating B cells. We found that cRel is heterogeneously distributed among naïve B cells, which are enriched for high expressors in a heavy-tailed distribution. We found that high cRel expressors show faster activation of the proliferative program, but do not sustain it well, with population expansion decaying earlier. With a mathematical model of the molecular network, we showed that cRel heterogeneity arises from balancing positive feedback by autoregulation and negative feedback by its inhibitor IκBε, confirmed by mouse knockouts. Using live-cell fluorescence microscopy, we showed that increased cRel primes B cells for early proliferation via higher basal expression of the cell cycle driver cMyc. However, peak cMyc induction amplitude is constrained by incoherent feedforward regulation, decoding the fold change of cRel activity to terminate the proliferative burst. This results in a complex nonlinear, nonmonotonic relationship between cRel expression and the extent of proliferation. These findings emphasize the importance of direct observational studies to complement gene knockout results and to learn about quantitative relationships between biological processes and their key regulators in the context of natural variations.
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Linfocitos B , Proliferación Celular , FN-kappa B , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Ratones , FN-kappa B/metabolismo , Ratones Noqueados , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-rel/metabolismo , Proteínas Proto-Oncogénicas c-rel/genéticaRESUMEN
The outcome of cancer and autoimmunity is often dictated by the effector functions of CD4+ conventional T cells (Tconv). Although activation of the NF-κB signaling pathway has long been implicated in Tconv biology, the cell-autonomous roles of the separate NF-κB transcription-factor subunits are unknown. Here, we dissected the contributions of the canonical NF-κB subunits RelA and c-Rel to Tconv function. RelA, rather than c-Rel, regulated Tconv activation and cytokine production at steady-state and was required for polarization toward the TH17 lineage in vitro. Accordingly, RelA-deficient mice were fully protected against neuroinflammation in a model of multiple sclerosis due to defective transition to a pathogenic TH17 gene-expression program. Conversely, Tconv-restricted ablation of c-Rel impaired their function in the microenvironment of transplanted tumors, resulting in enhanced cancer burden. Moreover, Tconv required c-Rel for the response to PD-1-blockade therapy. Our data reveal distinct roles for canonical NF-κB subunits in different disease contexts, paving the way for subunit-targeted immunotherapies.
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Esclerosis Múltiple , Neoplasias , Animales , Ratones , Linfocitos T CD4-Positivos , FN-kappa B , Transducción de Señal , Microambiente Tumoral , Proteínas Proto-Oncogénicas c-rel/metabolismoRESUMEN
Individual cell sensing of external cues has evolved through the temporal patterns in signaling. Since nuclear factor κB (NF-κB) signaling dynamics have been examined using a single subunit, RelA, it remains unclear whether more information might be transmitted via other subunits. Using NF-κB double-knockin reporter mice, we monitored both canonical NF-κB subunits, RelA and c-Rel, simultaneously in single macrophages by quantitative live-cell imaging. We show that signaling features of RelA and c-Rel convey more information about the stimuli than those of either subunit alone. Machine learning is used to predict the ligand identity accurately based on RelA and c-Rel signaling features without considering the co-activated factors. Ligand discrimination is achieved through selective non-redundancy of RelA and c-Rel signaling dynamics, as well as their temporal coordination. These results suggest a potential role of c-Rel in fine-tuning immune responses and highlight the need for approaches that will elucidate the mechanisms regulating NF-κB subunit specificity.
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FN-kappa B , Proteínas Proto-Oncogénicas c-rel , Ratones , Animales , FN-kappa B/metabolismo , Ligandos , Proteínas Proto-Oncogénicas c-rel/metabolismo , Factor de Transcripción ReIA/metabolismo , Transducción de Señal , Macrófagos/metabolismoRESUMEN
Although Parkinson's disease (PD) key neuropathological hallmarks are well known, the underlying pathogenic mechanisms of the disease still need to be elucidated to identify innovative disease-modifying drugs and specific biomarkers. NF-κB transcription factors are involved in regulating several processes associated with neurodegeneration, such as neuroinflammation and cell death, that could be related to PD pathology. NF-κB/c-Rel deficient (c-rel-/-) mice develop a progressive PD-like phenotype. The c-rel-/- mice present both prodromal and motor symptoms as well as key neuropathological features, including nigrostriatal dopaminergic neurons degeneration, accumulation of pro-apoptotic NF-κB/RelA acetylated at the lysine 310 residue (Ac-RelA(lys310)) and progressive caudo-rostral brain deposition of alpha-synuclein. c-Rel inhibition can exacerbate MPTP-induced neurotoxicity in mice. These findings support the claim that misregulation of c-Rel protein may be implicated in PD pathophysiology. In this study, we aimed at evaluating c-Rel levels and DNA-binding activity in human brains and peripheral blood mononuclear cells (PBMCs) of sporadic PD patients. We analyzed c-Rel protein content and activity in frozen substantia nigra (SN) samples from post-mortem brains of 10 PD patients and 9 age-matched controls as well as in PBMCs from 72 PD patients and 40 age-matched controls. c-Rel DNA-binding was significantly lower and inversely correlated with Ac-RelA(lys310) content in post-mortem SN of sporadic PD cases, when compared to healthy controls. c-Rel DNA-binding activity was also reduced in PBMCs of followed-up PD subjects. The decrease of c-Rel activity in PBMCs from PD patients appeared to be independent from dopaminergic medication or disease progression, as it was evident even in early stage, drug-naïve patients. Remarkably, the levels of c-Rel protein were comparable in PD and control subjects, pointing out a putative role for post-translational modifications of the protein in c-Rel dysfunctions. These findings support that PD is characterized by the loss of NF-κB/c-Rel activity that potentially has a role in PD pathophysiology. Future studies will be aimed at addressing whether the reduction of c-Rel DNA-binding could constitute a novel biomarker for PD.
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Intoxicación por MPTP , Enfermedad de Parkinson , Humanos , Ratones , Animales , FN-kappa B/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Leucocitos Mononucleares/metabolismo , Sustancia Negra/metabolismo , Neuronas Dopaminérgicas/metabolismo , Intoxicación por MPTP/patologíaRESUMEN
BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. The NF-κB transcription factor family subunit c-Rel is typically protumorigenic; however, it has recently been reported as a tumor suppressor. Here, we investigated the role of c-Rel in HCC. APPROACH AND RESULTS: Histological and transcriptional studies confirmed expression of c-Rel in human patients with HCC, but low c-Rel expression correlated with increased tumor cell proliferation and mutational burden and was associated with advanced disease. In vivo , global ( Rel-/- ) and epithelial specific ( RelAlb ) c-Rel knockout mice develop more tumors, with a higher proliferative rate and increased DNA damage, than wild-type (WT) controls 30 weeks after N-diethylnitrosamine injury. However, tumor burden was comparable when c-Rel was deleted in hepatocytes once tumors were established, suggesting c-Rel signaling is important for preventing HCC initiation after genotoxic injury, rather than for HCC progression. In vitro , Rel-/- hepatocytes were more susceptible to genotoxic injury than WT controls. ATM-CHK2 DNA damage response pathway proteins were suppressed in Rel-/- hepatocytes following genotoxic injury, suggesting that c-Rel is required for effective DNA repair. To determine if c-Rel inhibition sensitizes cancer cells to chemotherapy, by preventing repair of chemotherapy-induced DNA damage, thus increasing tumor cell death, we administered single or combination doxorubicin and IT-603 (c-Rel inhibitor) therapy in an orthotopic HCC model. Indeed, combination therapy was more efficacious than doxorubicin alone. CONCLUSION: Hepatocyte c-Rel signaling limits genotoxic injury and subsequent HCC burden. Inhibiting c-Rel as an adjuvant therapy increased the effectiveness of DNA damaging agents and reduced HCC growth.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Humanos , Ratones , Carcinogénesis/genética , Carcinoma Hepatocelular/patología , Daño del ADN , Doxorrubicina/farmacología , Hepatocitos/metabolismo , Neoplasias Hepáticas/metabolismo , Ratones Noqueados , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismoRESUMEN
Most of the currently known heterozygous pathogenic NFKB1 (Nuclear factor kappa B subunit 1) variants comprise deleterious defects such as severe truncations, internal deletions, and frameshift variants. Collectively, these represent the most frequent monogenic cause of common variable immunodeficiency (CVID) identified so far. NFKB1 encodes the transcription factor precursor p105 which undergoes limited proteasomal processing of its C-terminal half to generate the mature NF-κB subunit p50. Whereas p105/p50 haploinsufficiency due to devastating genetic damages and protein loss is a well-known disease mechanism, the pathogenic significance of numerous NFKB1 missense variants still remains uncertain and/or unexplored, due to the unavailability of accurate test procedures to confirm causality. In this study we functionally characterized 47 distinct missense variants residing within the N-terminal domains, thus affecting both proteins, the p105 precursor and the processed p50. Following transient overexpression of EGFP-fused mutant p105 and p50 in HEK293T cells, we used fluorescence microscopy, Western blotting, electrophoretic mobility shift assays (EMSA), and reporter assays to analyze their effects on subcellular localization, protein stability and precursor processing, DNA binding, and on the RelA-dependent target promoter activation, respectively. We found nine missense variants to cause harmful damage with intensified protein decay, while two variants left protein stability unaffected but caused a loss of the DNA-binding activity. Seven of the analyzed single amino acid changes caused ambiguous protein defects and four variants were associated with only minor adverse effects. For 25 variants, test results were indistinguishable from those of the wildtype controls, hence, their pathogenic impact remained elusive. In summary, we show that pathogenic missense variants affecting the Rel-homology domain may cause protein-decaying defects, thus resembling the disease-mechanisms of p105/p50 haploinsufficiency or may cause DNA-binding deficiency. However, rare variants (with a population frequency of less than 0.01%) with minor abnormalities or with neutral tests should still be considered as potentially pathogenic, until suitable tests have approved them being benign.
Asunto(s)
Mutación Missense , FN-kappa B , ADN , Células HEK293 , Humanos , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-rel/metabolismoRESUMEN
Type I interferon (IFN-I) is a key component of the host innate immune system. To establish efficient replication, viruses have developed several strategies to escape from the host IFN response. Japanese encephalitis virus (JEV) NS1', a larger NS1-related protein, is known to inhibit the mitochondrial antiviral signaling (MAVS)-mediated IFN-ß induction by increasing the binding of transcription factors (CREB and c-Rel) to the microRNA 22 (miRNA-22) promoter. However, the mechanism by which NS1' induces the recruitment of CREB and c-Rel onto the miRNA-22 promoter is unknown. Here, we found that JEV NS1' protein interacts with the host cyclin-dependent kinase 1 (CDK1) protein. Mechanistically, NS1' interrupts the CDC25C phosphatase-mediated dephosphorylation of CDK1, which prolongs the phosphorylation status of CDK1 and leads to the inhibition of MAVS-mediated IFN-ß induction. Furthermore, the CREB phosphorylation and c-Rel activation through the IκBα phosphorylation were observed to be enhanced upon the augmentation of CDK1 phosphorylation by NS1'. The abrogation of CDK1 activity by a small-molecule inhibitor significantly suppressed the JEV replication in vitro and in vivo. Moreover, the administration of CDK1 inhibitor protected the wild-type mice from JEV-induced lethality but showed no effect on the MAVS-/- mice challenged with JEV. In conclusion, our study provides new insight into the mechanism of JEV immune evasion, which may lead to the development of novel therapeutic options to treat JEV infection. IMPORTANCE Japanese encephalitis virus (JEV) is the main cause of acute human encephalitis in Asia. The unavailability of specific treatment for Japanese encephalitis demands a better understanding of the basic cellular mechanisms that contribute to the onset of disease. The present study identifies a novel interaction between the JEV NS1' protein and the cellular CDK1 protein, which facilitates the JEV replication by dampening the cellular antiviral response. This study sheds light on a novel mechanism of JEV replication, and thus our findings could be employed for developing new therapies against JEV infection.
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Proteína Quinasa CDC2/metabolismo , Virus de la Encefalitis Japonesa (Especie)/inmunología , Evasión Inmune/inmunología , Interferón beta/inmunología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína de Unión a CREB/metabolismo , Línea Celular Tumoral , Cricetinae , Encefalitis Japonesa/inmunología , Células HeLa , Humanos , Inmunidad Innata/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Inhibidor NF-kappaB alfa/metabolismo , Fosforilación/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Fosfatasas cdc25/metabolismoRESUMEN
Herpes simplex virus type 1 (HSV-1) is the predominant cause of herpes simplex encephalitis (HSE), a condition characterized by acute inflammation and viral replication in the brain. Host genetics contribute to HSE onset, including monogenic defects in type I interferon signaling in cases of childhood HSE. Mouse models suggest a further contribution of immune cell-mediated inflammation to HSE pathogenesis. We have previously described a truncating mutation in the c-Rel transcription factor (RelC307X) that drives lethal HSE in 60% of HSV-1-infected RelC307X mice. In this study, we combined dual host-virus RNA sequencing with flow cytometry to explore cell populations and mechanisms involved in RelC307X-driven HSE. At day 5 postinfection, prior to HSE clinical symptom onset, elevated HSV-1 transcription was detected together with augmented host interferon-stimulated and inflammatory gene expression in the brainstems of high-responding RelC307X mice, predictive of HSE development. This early induction of host gene expression preceded pathological infiltration of myeloid and T cells in RelC307X mice at HSE onset by day 7. Thus, we establish c-Rel as an early regulator of viral and host responses during mouse HSE. These data further highlight the importance of achieving a balanced immune response and avoiding excess interferon-driven inflammation to promote HSE resistance.
Asunto(s)
Encefalitis por Herpes Simple/metabolismo , Interferón Tipo I/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Animales , Encefalitis por Herpes Simple/virología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas Proto-Oncogénicas c-rel/genética , Transducción de Señal , Simplexvirus/genética , Simplexvirus/patogenicidad , Simplexvirus/fisiología , Linfocitos T/metabolismo , Linfocitos T/virologíaRESUMEN
Group 2 innate lymphoid cells (ILC2s) are emerging as important cellular regulators of homeostatic and disease-associated immune processes. The cytokine interleukin-33 (IL-33) promotes ILC2-dependent inflammation and immunity, with IL-33 having been shown to activate NF-κB in a wide variety of cell types. However, it is currently unclear which NF-κB members play an important role in IL-33-dependent ILC2 biology. Here, we identify the NF-κB family member c-Rel as a critical component of the IL-33-dependent activation of ILC2s. Although c-Rel is dispensable for ILC2 development, it is critical for ILC2 function in the lung, with c-Rel-deficient (c-Rel-/- ) mice present a significantly reduced response to papain- and IL-33-induced lung inflammation. We also show that the absence of c-Rel reduces the IL-33-dependent expansion of ILC2 precursors and lower levels of IL-5 and IL-13 cytokine production by mature ILC2s in the lung. Together, these results identify the IL-33-c-Rel axis as a central control point of ILC2 activation and function.
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Inmunidad Innata/efectos de los fármacos , Interleucina-33/farmacología , Pulmón/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Neumonía/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Médula Ósea/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Interleucina-13/metabolismo , Interleucina-5/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Papaína , Neumonía/inducido químicamente , Neumonía/genética , Neumonía/inmunología , Proteínas Proto-Oncogénicas c-rel/genéticaAsunto(s)
Integrasas/genética , Herencia Paterna , Recombinación Genética , Factores de Transcripción/genética , Animales , Femenino , Genotipo , Humanos , Integrasas/metabolismo , Masculino , Ratones Transgénicos , Mutación , Fenotipo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-rel/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Factor de Transcripción ReIB/genética , Factor de Transcripción ReIB/metabolismoRESUMEN
Natural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. We found that germline deficiency of NF-κB c-Rel results in a marked decrease in cytotoxic function of NK cells, both in vitro and in vivo, with no significant differences in the stages of NK cell development. We found that c-Rel binds to the promoters of perforin and granzyme B, two key proteins required for NK cytotoxicity, and controls their expression. We generated a NK cell specific c-Rel conditional knockout to study NK cell intrinsic role of c- Rel and found that both global and conditional c-Rel deficiency leads to decreased perforin and granzyme B expression and thereby cytotoxic function. We also confirmed the role of c-Rel in perforin and granzyme B expression in human NK cells. c-Rel reconstitution rescued perforin and granzyme B expressions in c-Rel deficient NK cells and restored their cytotoxic function. Our results show a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expressions and control of NK cell cytotoxic function.
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Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Animales , Células Cultivadas , Citocinas/biosíntesis , Citotoxicidad Inmunológica/genética , Granzimas/metabolismo , Humanos , Melanoma Experimental , Ratones , Ratones Noqueados , Modelos Biológicos , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Proto-Oncogénicas c-rel/genéticaRESUMEN
Sorting nexin 10 (SNX10) has been reported as a critical regulator in macrophage function, and germline SNX10 knockout effectively alleviated mouse colitis. Here, we investigated the precise role of SNX10 in inflammatory responses in macrophages in mouse colitis, and explored the druggability of SNX10 as a therapeutic target for inflammatory bowel disease (IBD). Our results revealed that myeloid-specific SNX10 deletion alleviated inflammation and pathological damage induced by dextran sulfate sodium (DSS). In vitro experiments showed that SNX10 deletion contributed to inflammation elimination by inhibiting PIKfyve-mediated TANK-binding kinase 1 (TBK1) /c-Rel signaling activation. Further study provided rational mechanism that SNX10 was required for the recruitment of PIKfyve to the TRIF-positive endosomes, through which PIKfyve activated TBK1/c-Rel for LPS-induced inflammation response. Based on the structure of SNX10, we discovered a new small-molecule inhibitor DC-SX029, which targeted SNX10 to block the SNX10-PIKfyve interaction, thereby decreased the TBK1/c-Rel signaling activation. Additionally, therapeutic efficiency of DC-SX029 was evaluated in both DSS-induced and IL10-deficient mouse colitis models. Our data demonstrate a new mechanism by which SNX10-PIKfyve interaction regulates LPS-induced inflammation response in macrophages via the TBK1/c-Rel signaling pathway. In vivo and in vitro pharmacological studies of SNX10 protein-protein interaction (PPI) inhibitor DC-SX029 demonstrate the feasibility of targeting SNX10 in IBD treatment.
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Antiinflamatorios/uso terapéutico , Colitis/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Transducción de Señal/efectos de los fármacos , Nexinas de Clasificación/efectos de los fármacos , Animales , Colitis/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nexinas de Clasificación/metabolismoRESUMEN
The NF-κB transcription factor c-Rel is a critical regulator of Treg ontogeny, controlling multiple points of the stepwise developmental pathway. Here, we found that the thymic Treg defect in c-Rel-deficient (cRel-/- ) mice is quantitative, not qualitative, based on analyses of TCR repertoire and TCR signaling strength. However, these parameters are altered in the thymic Treg-precursor population, which is also markedly diminished in cRel-/- mice. Moreover, c-Rel governs the transcriptional programme of both thymic and peripheral Tregs, controlling a core of genes involved with immune signaling, and separately in the periphery, cell cycle progression. Last, the immune suppressive function of peripheral cRel-/- tTregs is diminished in a lymphopenic model of T cell proliferation and is associated with decreased stability of Foxp3 expression. Collectively, we show that c-Rel is a transcriptional regulator that controls multiple aspects of Treg development, differentiation, and function via distinct mechanisms.
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Proteínas Proto-Oncogénicas c-rel/inmunología , Proteínas Proto-Oncogénicas c-rel/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Timo/inmunología , Timo/metabolismoRESUMEN
Tripartite motif-containing 9 (TRIM9) has been demonstrated to exert important roles in regulation of innate immune signaling. In this study, a novel TRIM9 homolog was identified from Penaeus monodon (named PmTRIM9). The open reading frame (ORF) of PmTRIM9 was 2064 bp, which encoding a 687-amino-acid polypeptide. Following Vibrio parahaemolyticus challenge, the expression levels of PmTRIM9 mRNA were significantly down-regulated in tested tissues. RNA interference and recombinant protein injection experiments were performed to explore the function of PmTRIM9, and the results showed it could facilitate V. parahaemolyticus replication and lead P. monodon more vulnerable to V. parahaemolyticus challenge. The dual-luciferase reporter assay showed that PmTRIM9 possessed the ability to inhibit the promoter activity in HEK293 T cells. Silencing of PmTRIM9 could increase the expression of the major NF-κB transcription factor, PmRelish. Further studies showed that knockdown of PmRelish promoted the V. parahaemolyticus infection and decreased the expression of specific antimicrobial peptides (AMPs), including PmCRU5, PmCRU7, PmALF6, PmALF3, PmLYZ and PmPEN5. However, knockdown of PmTRIM9 increased expression levels of the same AMPs, but except for PmCRU5, indicating that PmTRIM9 may negatively regulate the PmRelish-mediated expression of AMPs. All these results suggest that PmTRIM9 was involved in facilitating V. parahaemolyticus infection by inhibition of Relish pathway in P. monodon.
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Proteínas de Peces/genética , Penaeidae/microbiología , Proteínas Proto-Oncogénicas c-rel/antagonistas & inhibidores , Proteínas de Motivos Tripartitos/genética , Vibrio parahaemolyticus/crecimiento & desarrollo , Animales , Línea Celular , Silenciador del Gen , Células HEK293 , Humanos , Penaeidae/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Transducción de Señal/inmunologíaRESUMEN
O-GlcNAcylation is a reversible post-translational protein modification that regulates fundamental cellular processes including immune responses and autoimmunity. Previously, we showed that hyperglycemia increases O-GlcNAcylation of the transcription factor, nuclear factor kappaB c-Rel at serine residue 350 and enhances the transcription of the c-Rel-dependent proautoimmune cytokines interleukin-2, interferon gamma and granulocyte macrophage colony stimulating factor in T cells. c-Rel also plays a critical role in the transcriptional regulation of forkhead box P3 (FOXP3)-the master transcription factor that governs development and function of Treg cells. Here we show that the regulatory effect of c-Rel O-GlcNAcylation is gene-dependent, and in contrast to its role in enhancing the expression of proautoimmune cytokines, it suppresses the expression of FOXP3. Hyperglycemia-induced O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; streptozotocin-induced diabetes and spontaneous diabetes in nonobese diabetic mice. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases c-Rel binding at the FOXP3 promoter and negatively regulates FOXP3 expression. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating immunosuppressive FOXP3 expression and proautoimmune gene expression in autoimmune diabetes with potential therapeutic implications.
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Diabetes Mellitus Experimental , Factores de Transcripción Forkhead , Proteínas Proto-Oncogénicas c-rel , Animales , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Ratones , Proteínas Proto-Oncogénicas c-rel/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Proteínas Proto-Oncogénicas c-rel/farmacología , Linfocitos T ReguladoresRESUMEN
BACKGROUND: The structure of human galectin-16 (Gal-16) has yet to be solved, and its function has remained elusive. METHODS: X-ray crystallography was used to determine the atomic structures of Gal-16 and two of its mutants. The Gal-16 oligomer state was investigated by gel filtration, its hemagglutination activity was determined along with its ability to bind lactose using ITC. The cellular distribution of EGFP-tagged Gal-16 in various cell lines was also investigated, and the interaction between Gal-16 and c-Rel was assessed by pull-down studies, microscale thermophoresis and immunofluorescence. RESULTS: Unlike other galectins, Gal-16 lacks the ability to bind the ß-galactoside lactose. Lactose binding could be regained by replacing an arginine (Arg55) with asparagine, as shown in the crystal structures of two lactose-loaded Gal-16 mutants (R55N and R55N/H57R). Gal-16 was also shown to be monomeric by gel filtration, as well as in crystal structures. Thus, this galectin could not induce erythrocyte agglutination. EGFP-tagged Gal-16 was found to be localized mostly in the nucleus of various cell types, and can interact with c-Rel, a member of NF-κB family. CONCLUSIONS: Gal-16 exists as a monomer and its ligand binding is significantly different from that of other prototype galectins, suggesting that it has a novel function(s). The interaction between Gal-16 and c-Rel indicates that Gal-16 may regulate signal transduction pathways via the c-Rel hub in B or T cells at the maternal-fetal interface. GENERAL SIGNIFICANCE: The present study lays the foundation for further studies into the cellular and physiological functions of Gal-16.
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Lactosa/metabolismo , Linfocitos/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-ß1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.
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Epitelio/patología , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Macrófagos/patología , Proteínas Proto-Oncogénicas c-rel/genética , Animales , Polaridad Celular/genética , Marcación de Gen , Hepatocitos/patología , Hidroxiprolina/metabolismo , Cirrosis Hepática/prevención & control , Regeneración Hepática/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitosis/genética , Comunicación Paracrina/genética , Fosfofructoquinasa-2/genética , Proteínas Proto-Oncogénicas c-rel/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-rel/metabolismoRESUMEN
Genome-wide association studies have established that human REL is a susceptibility gene for lymphoid cancers and inflammatory diseases. REL is the hematopoietic member of the nuclear factor-κB (NF-κB) family and is frequently amplified in human lymphomas. However, the mechanism through which REL and its encoded protein c-Rel affect human lymphoma is largely unknown. Using both loss-of-function and gain-of-function approaches, we studied the roles of REL gene in human Jurkat leukemia cells. Compared with control Jurkat cells, REL knockout cells exhibited significant defects in cell growth and mitochondrial respiration. Genome-wide transcriptome analyses revealed that T cells lacking c-Rel had selective defects in the expression of inflammatory and metabolic genes including c-Myc. We found that c-Rel controlled the expression of c-Myc through its promotor, and expressing c-Myc in c-Rel-deficient lymphoma cells rescued their proliferative and metabolic defects. Thus, the human c-Rel-c-Myc axis controls lymphoma growth and metabolism and could be a therapeutic target for lymphomas.
Asunto(s)
Proliferación Celular/fisiología , Leucemia de Células T/metabolismo , Leucemia de Células T/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Técnicas de Inactivación de Genes , Humanos , Células JurkatRESUMEN
OBJECTIVES: NF-κB regulates genes that control inflammation, cell proliferation, differentiation and survival. Dysregulated NF-κB signalling alters normal skin physiology and deletion of cRel limits bleomycin-induced skin fibrosis. This study investigates the role of cRel in modulating fibroblast phenotype in the context of SSc. METHODS: Fibrosis was assessed histologically in mice challenged with bleomycin to induce lung or skin fibrosis. RNA sequencing and pathway analysis was performed on wild type and Rel-/- murine lung and dermal fibroblasts. Functional assays examined fibroblast proliferation, migration and matrix production. cRel overexpression was investigated in human dermal fibroblasts. cRel immunostaining was performed on lung and skin tissue sections from SSc patients and non-fibrotic controls. RESULTS: cRel expression was elevated in murine lung and skin fibrosis models. Rel-/- mice were protected from developing pulmonary fibrosis. Soluble collagen production was significantly decreased in fibroblasts lacking cRel while proliferation and migration of these cells was significantly increased. cRel regulates genes involved in extracellular structure and matrix organization. Positive cRel staining was observed in fibroblasts in human SSc skin and lung tissue. Overexpression of constitutively active cRel in human dermal fibroblasts increased expression of matrix genes. An NF-κB gene signature was identified in diffuse SSc skin and nuclear cRel expression was elevated in SSc skin fibroblasts. CONCLUSION: cRel regulates a pro-fibrogenic transcriptional programme in fibroblasts that may contribute to disease pathology. Targeting cRel signalling in fibroblasts of SSc patients could provide a novel therapeutic avenue to limit scar formation in this disease.