RESUMO
Human inborn errors of thymic T cell tolerance underlie the production of autoantibodies (auto-Abs) neutralizing type I IFNs, which predispose to severe viral diseases. We analyze 131 female patients with X-linked dominant incontinentia pigmenti (IP), heterozygous for loss-of-function (LOF) NEMO variants, from 99 kindreds in 10 countries. Forty-seven of these patients (36%) have auto-Abs neutralizing IFN-α and/or IFN-ω, a proportion 23 times higher than that for age-matched female controls. This proportion remains stable from the age of 6 years onward. On imaging, female patients with IP have a small, abnormally structured thymus. Auto-Abs against type I IFNs confer a predisposition to life-threatening viral diseases. By contrast, patients with IP lacking auto-Abs against type I IFNs are at no particular risk of viral disease. These results suggest that IP accelerates thymic involution, thereby underlying the production of auto-Abs neutralizing type I IFNs in at least a third of female patients with IP, predisposing them to life-threatening viral diseases.
Assuntos
Autoanticorpos , Quinase I-kappa B , Incontinência Pigmentar , Interferon Tipo I , Timo , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Feminino , Autoanticorpos/imunologia , Timo/imunologia , Timo/patologia , Criança , Incontinência Pigmentar/imunologia , Incontinência Pigmentar/genética , Incontinência Pigmentar/patologia , Pré-Escolar , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Viroses/imunologia , Lactente , Adulto , Adolescente , Adulto JovemRESUMO
PURPOSE: Genetic hypomorphic defects in X chromosomal IKBKG coding for the NF-κB essential modulator (NEMO) lead to ectodermal dysplasia and immunodeficiency in males and the skin disorder incontinentia pigmenti (IP) in females, respectively. NF-κB essential modulator (NEMO) Δ-exon 5-autoinflammatory syndrome (NEMO-NDAS) is a systemic autoinflammatory disease caused by alternative splicing and increased proportion of NEMO-Δex5. We investigated a female carrier presenting with IP and NEMO-NDAS due to non-skewed X-inactivation. METHODS: IKBKG transcripts were quantified in peripheral blood mononuclear cells isolated from the patient, her mother, and healthy controls using RT-PCR and nanopore sequencing. Corresponding proteins were analyzed by western blotting and flow cytometry. Besides toll-like receptor (TLR) and tumor necrosis factor (TNF) signaling, the interferon signature, cytokine production and X-inactivation status were investigated. RESULTS: IP and autoinflammation with recurrent fever, oral ulcers, hepatitis, and neutropenia, but no immunodeficiency was observed in a female patient. Besides moderately reduced NEMO signaling function, type I interferonopathy, and elevated IL-18 and CXCL10 were found. She and her mother both carried the heterozygous variant c.613 C > T p.(Gln205*) in exon 5 of IKBKG previously reported in NEMO-deficient patients. However, X-inactivation was skewed in the mother, but not in the patient. Alternative splicing led to increased ratios of NEMO-Dex5 over full-length protein in peripheral blood cell subsets causing autoinflammation. Clinical symptoms partially resolved under treatment with TNF inhibitors. CONCLUSION: Non-skewed X-inactivation can lead to NEMO-NDAS in females with IP carrying hypomorphic IKBKG variants due to alternative splicing and increased proportions of NEMO-∆ex5.
Assuntos
Éxons , Quinase I-kappa B , Incontinência Pigmentar , Inativação do Cromossomo X , Humanos , Feminino , Incontinência Pigmentar/genética , Incontinência Pigmentar/diagnóstico , Quinase I-kappa B/genética , Éxons/genética , Doenças Hereditárias Autoinflamatórias/genética , Doenças Hereditárias Autoinflamatórias/diagnóstico , Mutação/genética , Citocinas/metabolismo , Adulto , Processamento Alternativo , Transdução de SinaisRESUMO
Gastrointestinal cancers account for over a quarter of all cancer cases and are associated with poor prognosis and high mortality rates. The IKK complex (the canonical I kappa B kinase), comprising the CHUK, IKBKB, and IKBKG genes, plays a crucial role in activating the NF-kB signaling pathway. This study aimed to analyze publicly available bioinformatics data to elucidate the oncogenic role of IKK genes in selected gastrointestinal cancers. Our findings reveal that IKBKB and IKBKG are significantly upregulated in all examined cancers, while CHUK is upregulated in esophageal carcinoma and stomach adenocarcinoma. Additionally, the expression of IKK genes varies with histological grade and nodal metastases. For instance, in stomach adenocarcinoma, CHUK and IKBKB are upregulated in higher histological grades and greater lymph node infiltration. Lower expression levels of CHUK, IKBKB, and IKBKG in stomach adenocarcinoma and IKBKB in esophageal squamous cell carcinoma correlate with shorter overall survival. Conversely, in esophageal adenocarcinoma, reduced IKBKG expression is linked to longer overall survival, while higher IKBKB expression in colon adenocarcinoma is associated with longer overall survival. Given the significant role of IKK genes in the development and progression of selected gastrointestinal cancers, they hold potential as prognostic markers and therapeutic targets, offering valuable insights for clinical practice.
Assuntos
Biologia Computacional , Neoplasias Gastrointestinais , Regulação Neoplásica da Expressão Gênica , Quinase I-kappa B , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Biologia Computacional/métodos , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Neoplasias Gastrointestinais/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidade , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Prognóstico , Biomarcadores Tumorais/genética , Transdução de Sinais/genéticaRESUMO
The regulatory role of the inhibitor of NF-kB kinase ε (IKKε) in postmyocardial infarction (MI) inflammation remains uncertain. Using an MI mouse model, we examined the cardiac outcomes of IKKε knockout (KO) mice and wild-type mice. We employed single-cell RNA sequencing (scRNA-seq) and phosphorylated protein array techniques to profile cardiac macrophages. IKKε KO mice exhibited compromised survival, heightened inflammation, pronounced cardiac fibrosis, and a reduced ejection fraction. A distinct cardiac macrophage subset in IKKε KO mice exhibited increased fibrotic marker expression and decreased phosphorylated p38 (p-p38) levels, indicating an enhanced macrophage-myofibroblast transition (MMT) post-MI. While cardiac inflammation is crucial for initiating compensatory pathways, the timely resolution of inflammation was impaired in the IKKε KO group, while the MMT in macrophages accelerated post-MI, leading to cardiac failure. Additionally, our study highlighted the potential of 5-azacytidine (5-Aza), known for its anti-inflammatory and cardioprotective effects, in restoring p-p38 levels in stimulated macrophages. The administration of 5-Aza significantly reduced the MMT in cardiac macrophages from the IKKε KO group. These findings underscore the regulation of the inflammatory response and macrophage transition by the IKKε-p38 axis, indicating that the MMT is a promising therapeutic target for ischemic heart disease.
Assuntos
Modelos Animais de Doenças , Quinase I-kappa B , Macrófagos , Camundongos Knockout , Infarto do Miocárdio , Miofibroblastos , Animais , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Macrófagos/metabolismo , Camundongos , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Miofibroblastos/metabolismo , Miofibroblastos/patologia , MasculinoRESUMO
The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKß and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBß) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/ß dimerization in substrate motif recognition.
Assuntos
Motivos de Aminoácidos , Quinase I-kappa B , NF-kappa B , Multimerização Proteica , Quinase I-kappa B/metabolismo , Quinase I-kappa B/química , Quinase I-kappa B/genética , Humanos , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Transdução de Sinais , Inibidor de NF-kappaB alfa/metabolismo , Inibidor de NF-kappaB alfa/genética , Simulação de Acoplamento Molecular , Células HEK293 , Especificidade por SubstratoRESUMO
Doxorubicin (DOX) is an effective chemotherapeutic drug, but its use can lead to cardiomyopathy, which is the leading cause of mortality among cancer patients. Macrophages play a role in DOX-induced cardiomyopathy (DCM), but the mechanisms undlerlying this relationship remain unclear. This study aimed to investigate how IKKα regulates macrophage activation and contributes to DCM in a mouse model. Specifically, the role of macrophage IKKα was evaluated in macrophage-specific IKKα knockout mice that received DOX injections. The findings revealed increased expression of IKKα in heart tissues after DOX administration. In mice lacking macrophage IKKα, myocardial injury, ventricular remodeling, inflammation, and proinflammatory macrophage activation worsened in response to DOX administration. Bone marrow transplant studies confirmed that IKKα deficiency exacerbated cardiac dysfunction. Macrophage IKKα knockout also led to mitochondrial damage and metabolic dysfunction in macrophages, thereby resulting in increased cardiomyocyte injury and oxidative stress. Single-cell sequencing analysis revealed that IKKα directly binds to STAT3, leading to the activation of STAT3 phosphorylation at S727. Interestingly, the inhibition of STAT3-S727 phosphorylation suppressed both DCM and cardiomyocyte injury. In conclusion, the IKKα-STAT3-S727 signaling pathway was found to play a crucial role in DOX-induced cardiomyopathy. Targeting this pathway could be a promising therapeutic strategy for treating DOX-related heart failure.
Assuntos
Cardiomiopatias , Doxorrubicina , Quinase I-kappa B , Macrófagos , Camundongos Knockout , Fator de Transcrição STAT3 , Transdução de Sinais , Animais , Doxorrubicina/efeitos adversos , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/genética , Camundongos , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Transdução de Sinais/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Modelos Animais de Doenças , Ativação de Macrófagos/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
While immune checkpoint blockade (ICB) has shown promise for clinical cancer therapy, its efficacy has only been observed in a limited subset of patients and the underlying mechanisms regulating innate and acquired resistance to ICB of tumor cells remain poorly understood. Here, we identified ependymin-related protein 1 (EPDR1) as an important tumor-intrinsic regulator of PD-L1 expression and tumor immune evasion. Aberrant expression of EPDR1 in hepatocellular carcinoma is associated with immunosuppression. Mechanistically, EPDR1 binds to E3 ligase TRIM21 and disrupts its interaction with IkappaB kinase-b, suppressing its ubiquitylation and autophagosomal degradation and enhancing NF-κB-mediated transcriptional activation of PD-L1. Further, we validated through a mouse liver cancer model that EPDR1 mediates exhaustion of CD8+ T cells and promotes tumor progression. In addition, we observed a positive correlation between EPDR1 and PD-L1 expression in both human and mouse liver cancer samples. Collectively, our study reveals a previously unappreciated role of EPDR1 in orchestrating tumor immune evasion and cancer progression.
Assuntos
Antígeno B7-H1 , Carcinoma Hepatocelular , Neoplasias Hepáticas , Evasão Tumoral , Ubiquitinação , Animais , Humanos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Camundongos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Proteínas com Domínio MARVEL/metabolismo , Proteínas com Domínio MARVEL/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Camundongos Endogâmicos C57BL , RibonucleoproteínasRESUMO
The IκB Kinase (IKK) complex, containing catalytic IKK2 and noncatalytic NEMO subunits, plays essential roles in the induction of transcription factors of the NF-κB family. Catalytic activation of IKK2 via phosphorylation of its activation loop is promoted upon noncovalent association of linear or K63-linked polyubiquitin chains to NEMO within the IKK complex. The mechanisms of this activation remain speculative. To investigate interaction dynamics within the IKK complex during activation of IKK2, we conducted hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on NEMO and IKK2 proteins in their free and complex-bound states. Altered proton exchange profiles were observed in both IKK2 and NEMO upon complex formation, and changes were consistent with the involvement of distinct regions throughout the entire length of both proteins, including previously uncharacterized segments, in direct or allosteric interactions. Association with linear tetraubiquitin (Ub4) affected multiple regions of the IKK2:NEMO complex, in addition to previously identified interaction sites on NEMO. Intriguingly, observed enhanced solvent accessibility of the IKK2 activation loop within the IKK2:NEMO:Ub4 complex, coupled with contrasting protection of surrounding segments of the catalytic subunit, suggests an allosteric role for NEMO:Ub4 in priming IKK2 for phosphorylation-dependent catalytic activation.
Assuntos
Quinase I-kappa B , Quinase I-kappa B/metabolismo , Quinase I-kappa B/química , Quinase I-kappa B/genética , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Ativação Enzimática , Fosforilação , Ubiquitina/metabolismo , Ubiquitina/química , Modelos Moleculares , Ligação ProteicaRESUMO
Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift and luciferase reporter assays. To validate the approach, we studied rs2297550 in detail, finding that the risk allele enhanced binding to the transcription factor Ikaros (encoded by IKZF1), thereby modulating expression of IKBKE. Correspondingly, primary cells from genotyped healthy donors bearing the risk allele expressed higher levels of the interferon / NF-κB regulator IKKε. Together, these findings define a set of likely functional non-coding lupus risk variants and identify a regulatory pathway involving rs2297550, Ikaros, and IKKε implicated by human genetics in risk for SLE.
Assuntos
Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Quinase I-kappa B , Fator de Transcrição Ikaros , Lúpus Eritematoso Sistêmico , Polimorfismo de Nucleotídeo Único , Lúpus Eritematoso Sistêmico/genética , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Predisposição Genética para Doença/genética , Alelos , Linfócitos B/metabolismo , NF-kappa B/metabolismo , NF-kappa B/genética , Regulação da Expressão GênicaRESUMO
TNF is a potent cytokine known for its involvement in physiology and pathology. In Rheumatoid Arthritis (RA), persistent TNF signals cause aberrant activation of synovial fibroblasts (SFs), the resident cells crucially involved in the inflammatory and destructive responses of the affected synovial membrane. However, the molecular switches that control the pathogenic activation of SFs remain poorly defined. Cyld is a major component of deubiquitination (DUB) machinery regulating the signaling responses towards survival/inflammation and programmed necrosis that induced by cytokines, growth factors and microbial products. Herein, we follow functional genetic approaches to understand how Cyld affects arthritogenic TNF signaling in SFs. We demonstrate that in spontaneous and induced RA models, SF-Cyld DUB deficiency deteriorates arthritic phenotypes due to increased levels of chemokines, adhesion receptors and bone-degrading enzymes generated by mutant SFs. Mechanistically, Cyld serves to restrict the TNF-induced hyperactivation of SFs by limiting Tak1-mediated signaling, and, therefore, leading to supervised NF-κB and JNK activity. However, Cyld is not critically involved in the regulation of TNF-induced death of SFs. Our results identify SF-Cyld as a regulator of TNF-mediated arthritis and inform the signaling landscape underpinning the SF responses.
Assuntos
Artrite Reumatoide , Enzima Desubiquitinante CYLD , Fibroblastos , Quinase I-kappa B , MAP Quinase Quinase Quinases , Transdução de Sinais , Membrana Sinovial , Fibroblastos/metabolismo , Fibroblastos/patologia , Enzima Desubiquitinante CYLD/metabolismo , Enzima Desubiquitinante CYLD/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , Animais , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Camundongos , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Humanos , NF-kappa B/metabolismo , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
IkappaB kinase beta (IKKß) is a key member of IκB kinases and functions importantly in interferon (IFN) signaling. Phosphorylation and ubiquitination are involved in the activation of IKKß. A20 is a de-ubiquitin enzyme and functions as a suppressor in inflammation signaling, which has been reported to be phosphorylated and activated by IKKß. However, the role and relationship of IKKß and A20 in teleost remains unclear. In this study, IKKß (bcIKKß) and A20 (bcA20) of black carp (Mylopharyngodon piceus) have been cloned and characterized. Overexpressed bcIKKß in EPC cells showed strong anti-viral ability by activating both NF-κB and IFN signaling. EPC cells stable expressing bcIKKß presented improved anti-viral activity as well. The interaction between bcA20 and bcIKKß was identified, and overexpression of bcA20 was able to suppress bcIKKß-mediated activation of NF-κB and IFN signaling. Meanwhile, knock-down of A20 increased host the antiviral ability of host cells. Importantly, it has been identified that bcA20 was able to remove K27-linked ubiquitination and decrease the phosphorylation of bcIKKß. Thus, our data conclude that bcA20 suppresses the anti-viral activity of bcIKKß and removes its K27-linked ubiquitination, which presents a new mechanism of IKKß regulation.
Assuntos
Carpas , Proteínas de Peixes , Quinase I-kappa B , Transdução de Sinais , Ubiquitinação , Animais , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , Carpas/imunologia , Carpas/genética , Transdução de Sinais/imunologia , Interferons/genética , Interferons/imunologia , Interferons/metabolismo , Doenças dos Peixes/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Imunidade Inata/genética , Regulação da Expressão Gênica/imunologia , Alinhamento de Sequência/veterinária , Filogenia , Perfilação da Expressão Gênica/veterinária , Sequência de AminoácidosRESUMO
In response to DNA double-strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm, where it promotes monoubiquitylation of Nemo and the resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as LPSs. To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy and fertile and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Quebras de DNA de Cadeia Dupla , NF-kappa B , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Camundongos , Fosforilação , NF-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Camundongos Knockout , Etoposídeo/farmacologia , Motivos de AminoácidosRESUMO
Pityriasis rubra pilaris (PRP) is an inflammatory papulosquamous dermatosis, characterized by hyperkeratotic follicular papules and erythematous desquamative plaques. The precise pathogenic mechanism underlying PRP remains incompletely understood. Herein, we conduct a case-control study involving a cohort of 102 patients with sporadic PRP and 800 healthy controls of Han Chinese population and identify significant associations (P = 1.73 × 10-6) between PRP and heterozygous mutations in the Keratin 32 gene (KRT32). KRT32 is found to be predominantly localized in basal keratinocytes and exhibits an inhibitory effect on skin inflammation by antagonizing the NF-κB pathway. Mechanistically, KRT32 binds to NEMO, promoting excessive K48-linked polyubiquitination and NEMO degradation, which hinders IKK complex formation. Conversely, loss-of-function mutations in KRT32 among PRP patients result in NF-κB hyperactivation. Importantly, Krt32 knockout mice exhibit a PRP-like dermatitis phenotype, suggesting compromised anti-inflammatory function of keratinocytes in response to external pro-inflammatory stimuli. This study proposes a role for KRT32 in regulating inflammatory immune responses, with damaging variants in KRT32 being an important driver in PRP development. These findings offer insights into the regulation of skin immune homeostasis by keratin and open up the possibility of using KRT32 as a therapeutic target for PRP.
Assuntos
Queratinócitos , Pitiríase Rubra Pilar , Pele , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto Jovem , Estudos de Casos e Controles , Homeostase , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinas/metabolismo , Queratinas/genética , Mutação com Perda de Função , Camundongos Knockout , NF-kappa B/metabolismo , Pitiríase Rubra Pilar/genética , Pitiríase Rubra Pilar/imunologia , Pitiríase Rubra Pilar/patologia , Pitiríase Rubra Pilar/metabolismo , Transdução de Sinais , Pele/patologia , Pele/imunologia , Pele/metabolismo , UbiquitinaçãoRESUMO
The aim of this study was to investigate the signaling pathways involved in the proliferation and differentiation of pig Sertoli cells (SCs) mediated by thyroid hormone (T3) to provide a theoretical and practical basis for enhancing pig semen production. The effects of different concentrations of T3 on the proliferation of pig SCs were evaluated using the CCK8 assay. The impact of T3 on the proliferation and differentiation of pig SCs was further examined using RNA-seq, qPCR, and Western Blotting techniques. Additionally, the involvement of the p38 MAPK and NFκB pathways in mediating the effects of T3 on SCs proliferation and differentiation was investigated. Our findings revealed a strong correlation between the dosage of T3 and the inhibition of pig SCs proliferation and promotion of maturation. T3 regulated the activation state of the NFκB signaling pathway by upregulating IKKα, downregulating IKKß, and promoting IκB phosphorylation. Furthermore, T3 facilitated SCs maturation by upregulating AR and FSHR expression while downregulating KRT-18. In conclusion, T3 inhibits pig SCs proliferation and promote pig SCs maturation through the IKK/NFκB and p38 MAPK pathways. These findings provide valuable insights into the mechanisms by which T3 influences the proliferation and maturation of pig SCs.
Assuntos
Proliferação de Células , NF-kappa B , Células de Sertoli , Transdução de Sinais , Tiroxina , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Masculino , Suínos , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiroxina/farmacologia , Linhagem Celular , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacosRESUMO
LAIR1, a receptor found on immune cells, is capable of binding to collagen and is involved in immune-related diseases. However, the precise contribution of LAIR1 expressed on hepatocellular carcinoma (HCC) cells to tumor microenvironment is still unclear. In our study, bioinformatics analysis and immunofluorescence were employed to study the correlation between LAIR1 levels and clinical indicators. Transwell and scratch tests were used to evaluate how LAIR1 affected the migration and invasion of HCC cells. The chemotactic capacity and alternative activation of macrophages were investigated using RT-qPCR, transwell, and immunofluorescence. To investigate the molecular mechanisms, transcriptome sequencing analysis, Western blot, nucleus/cytoplasm fractionation, ELISA, and cytokine microarray were employed. We revealed a significant correlation between the presence of LAIR1 and an unfavorable outcome in HCC. We indicated that LAIR1 promoted migration and invasion of HCC cells through the AKT-IKKß-p65 axis. Additionally, the alternative activation and infiltration of tumor-associated macrophages induced by LAIR1 were reliant on the upregulation of IL6 and CCL5 within this axis, respectively. In conclusion, blocking LAIR1 was found to be an effective approach in combating the cancerous advancement of HCC.
Assuntos
Carcinoma Hepatocelular , Movimento Celular , Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-akt , Receptores Imunológicos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Linhagem Celular Tumoral , Microambiente Tumoral , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais , Macrófagos/metabolismo , Macrófagos/patologia , Proliferação de Células , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia , Macrófagos Associados a Tumor/imunologia , Invasividade NeoplásicaRESUMO
IκB kinase (IKK)α controls noncanonical NF-κB signaling required for lymphoid organ development. We showed previously that lymph node formation is ablated in IkkαLyve-1 mice constitutively lacking IKKα in lymphatic endothelial cells (LECs). We now reveal that loss of IKKα in LECs leads to the formation of BALT in the lung. Tertiary lymphoid structures appear only in the lungs of IkkαLyve-1 mice and are not present in any other tissues, and these highly organized BALT structures form after birth and in the absence of inflammation. Additionally, we show that IkkαLyve-1 mice challenged with influenza A virus (IAV) exhibit markedly improved survival and reduced weight loss compared with littermate controls. Importantly, we determine that the improved morbidity and mortality of IkkαLyve-1 mice is independent of viral load and rate of clearance because both mice control and clear IAV infection similarly. Instead, we show that IFN-γ levels are decreased, and infiltration of CD8 T cells and monocytes into IkkαLyve-1 lungs is reduced. We conclude that ablating IKKα in LECs promotes BALT formation and reduces the susceptibility of IkkαLyve-1 mice to IAV infection through a decrease in proinflammatory stimuli.
Assuntos
Homeostase , Quinase I-kappa B , Vírus da Influenza A , Pulmão , Infecções por Orthomyxoviridae , Animais , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Camundongos , Pulmão/imunologia , Pulmão/virologia , Pulmão/patologia , Infecções por Orthomyxoviridae/imunologia , Vírus da Influenza A/imunologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Linfócitos T CD8-Positivos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologia , Interferon gama/metabolismoAssuntos
Testes Genéticos , Quinase I-kappa B , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/deficiência , Masculino , Feminino , MutaçãoRESUMO
Signal transduction proteins containing a pLxIS motif induce interferon (IFN) responses central to antiviral immunity. Apart from their established roles in activating the IFN regulator factor (IRF) transcription factors, the existence of additional pathways and functions associated with the pLxIS motif is unknown. Using a synthetic biology-based platform, we identified two orphan pLxIS-containing proteins that stimulate IFN responses independent of all known pattern-recognition receptor pathways. We further uncovered a diversity of pLxIS signaling mechanisms, where the pLxIS motif represents one component of a multi-motif signaling entity, which has variable functions in activating IRF3, the TRAF6 ubiquitin ligase, IκB kinases, mitogen-activated protein kinases, and metabolic activities. The most diverse pLxIS signaling mechanisms were associated with the highest antiviral activities in human cells. The flexibility of domains that regulate IFN signaling may explain their prevalence in nature.
Assuntos
Fator Regulador 3 de Interferon , Interferons , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Humanos , Interferons/metabolismo , Células HEK293 , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Domínios Proteicos , Animais , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Motivos de Aminoácidos , Proteínas Quinases Ativadas por Mitógeno/metabolismoRESUMO
While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1ß and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1ß- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1ß- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1ß may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.
Assuntos
Células Epiteliais , Quinase I-kappa B , Humanos , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células A549 , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Interleucina-1beta/farmacologia , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , NF-kappa B/metabolismo , NF-kappa B/genética , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Pulmão/metabolismo , Pulmão/citologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/citologia , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Exposure to excess ammonia-N (NH3/NH4+) in aquaculture can disrupt physiological function in shrimp leading to enhanced oxidative stress and apoptosis, but little is known concerning the post-transcriptional regulation mechanism. In this study, the first miR-200 family member in crustacean was identified and characterized from Litopenaeus vannamei (designed as Lva-miR-8-3p). Lva-miR-8-3p was highly expressed in eyestalks, brainganglion, and gills. The expression of Lva-miR-8-3p in gills significantly decreased after ammonia-N stress, and Lva-miR-8-3p was confirmed to target IKKß 3'UTR for negatively regulating IKKß/NF-κB pathway. Overexpression of miR-8-3p promoted the hemolymph ammonia-N accumulation, total hemocyte count (THC) decrease, and gills tissue damage, thus resulting in a decreased survival rate of ammonia-exposed shrimp. Besides, Lva-miR-8-3p silencing could enhance the antioxidant enzymes activities and reduce the oxidative damage, whereas overexpression of Lva-miR-8-3p exerted the opposite effects. Furthermore, Lva-miR-8-3p overexpression was found to aggravate ammonia-N induced apoptosis in gills. In primarily cultured hemocytes, the cell viability decreased, the ROS content and caspase-3 activity increased after agomiR-8-3p transfection, while antagomiR-8-3p transfection caused the opposite change except the cell viability. These findings indicate that Lva-miR-8-3p acts as a post-transcriptional regulator in ammonia-N induced antioxidant response and apoptosis by negatively regulating IKKß/NF-κB pathway.