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1.
EMBO J ; 39(1): e101533, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31701553

RESUMO

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 "master" enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interleucina-1alfa/farmacologia , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Sítios de Ligação , Células Cultivadas , Quimiocinas/metabolismo , Cromatina/química , Cromatina/genética , Células HeLa , Humanos , MAP Quinase Quinase Quinases/genética , NF-kappa B/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia
2.
Basic Res Cardiol ; 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38758338

RESUMO

The right ventricle (RV) differs developmentally, anatomically and functionally from the left ventricle (LV). Therefore, characteristics of LV adaptation to chronic pressure overload cannot easily be extrapolated to the RV. Mitochondrial abnormalities are considered a crucial contributor in heart failure (HF), but have never been compared directly between RV and LV tissues and cardiomyocytes. To identify ventricle-specific mitochondrial molecular and functional signatures, we established rat models with two slowly developing disease stages (compensated and decompensated) in response to pulmonary artery banding (PAB) or ascending aortic banding (AOB). Genome-wide transcriptomic and proteomic analyses were used to identify differentially expressed mitochondrial genes and proteins and were accompanied by a detailed characterization of mitochondrial function and morphology. Two clearly distinguishable disease stages, which culminated in a comparable systolic impairment of the respective ventricle, were observed. Mitochondrial respiration was similarly impaired at the decompensated stage, while respiratory chain activity or mitochondrial biogenesis were more severely deteriorated in the failing LV. Bioinformatics analyses of the RNA-seq. and proteomic data sets identified specifically deregulated mitochondrial components and pathways. Although the top regulated mitochondrial genes and proteins differed between the RV and LV, the overall changes in tissue and cardiomyocyte gene expression were highly similar. In conclusion, mitochondrial dysfuntion contributes to disease progression in right and left heart failure. Ventricle-specific differences in mitochondrial gene and protein expression are mostly related to the extent of observed changes, suggesting that despite developmental, anatomical and functional differences mitochondrial adaptations to chronic pressure overload are comparable in both ventricles.

3.
Mol Cell ; 62(6): 943-957, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-27315556

RESUMO

Signals and posttranslational modifications regulating the decapping step in mRNA degradation pathways are poorly defined. In this study we reveal the importance of K63-linked ubiquitylation for the assembly of decapping factors, P-body formation, and constitutive decay of instable mRNAs encoding mediators of inflammation by various experimental approaches. K63-branched ubiquitin chains also regulate IL-1-inducible phosphorylation of the P-body component DCP1a. The E3 ligase TRAF6 binds to DCP1a and indirectly regulates DCP1a phosphorylation, expression of decapping factors, and gene-specific mRNA decay. Mutation of six C-terminal lysines of DCP1a suppresses decapping activity and impairs the interaction with the mRNA decay factors DCP2, EDC4, and XRN1, but not EDC3, thus remodeling P-body architecture. The usage of ubiquitin chains for the proper assembly and function of the decay-competent mammalian decapping complex suggests an additional layer of control to allow a coordinated function of decapping activities and mRNA metabolism in higher eukaryotes.


Assuntos
Endorribonucleases/metabolismo , Lisina/metabolismo , Capuzes de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Transativadores/metabolismo , Ubiquitinação , Animais , Linhagem Celular Tumoral , Endorribonucleases/genética , Exorribonucleases/metabolismo , Células HEK293 , Humanos , Interleucina-1alfa/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas/metabolismo , Capuzes de RNA/genética , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , Receptores de Interleucina-1/agonistas , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fatores de Tempo , Transativadores/genética , Transfecção , Ubiquitinação/efeitos dos fármacos
4.
Mol Cell ; 53(2): 193-208, 2014 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-24389100

RESUMO

Given the intimate link between inflammation and dysregulated cell proliferation in cancer, we investigated cytokine-triggered gene expression in different cell cycle stages. Transcriptome analysis revealed that G1 release through cyclin-dependent kinase 6 (CDK6) and CDK4 primes and cooperates with the cytokine-driven gene response. CDK6 physically and functionally interacts with the NF-κB subunit p65 in the nucleus and is found at promoters of many transcriptionally active NF-κB target genes. CDK6 recruitment to distinct chromatin regions of inflammatory genes was essential for proper loading of p65 to its cognate binding sites and for the function of p65 coactivators, such as TRIP6. Furthermore, cytokine-inducible nuclear translocation and chromatin association of CDK6 depends on the kinase activity of TAK1 and p38. These results have widespread biological implications, as aberrant CDK6 expression or activation that is frequently observed in human tumors modulates NF-κB to shape the cytokine and chemokine repertoires in chronic inflammation and cancer.


Assuntos
Cromatina/metabolismo , Quinase 6 Dependente de Ciclina/fisiologia , NF-kappa B/genética , Ciclo Celular/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/fisiologia , Quinase 6 Dependente de Ciclina/análise , Quinase 6 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Interleucina-1/metabolismo , Interleucina-1/fisiologia , Interleucina-8/genética , Interleucina-8/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Regiões Promotoras Genéticas , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
5.
Nucleic Acids Res ; 48(7): 3496-3512, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32107550

RESUMO

Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.


Assuntos
Histona Desacetilases/metabolismo , Leucemia/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Humanos , Leucemia/enzimologia , Lisina/metabolismo , Camundongos , Mutação , Peptídeos Cíclicos/farmacologia , Estabilidade Proteica , Receptor Notch1/química , Receptor Notch1/genética
6.
J Allergy Clin Immunol ; 146(4): 694-705, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32771559

RESUMO

The nuclear factor kappa B (NF-κB) signaling system, a key regulator of immunologic processes, also affects a plethora of metabolic changes associated with inflammation and the immune response. NF-κB-regulating signaling cascades, in concert with NF-κB-mediated transcriptional events, control the metabolism at several levels. NF-κB modulates apical components of metabolic processes including metabolic hormones such as insulin and glucagon, the cellular master switches 5' AMP-activated protein kinase and mTOR, and also numerous metabolic enzymes and their respective regulators. Vice versa, metabolic enzymes and their products also exert multilevel control of NF-κB activity, thereby creating a highly connected regulatory network. These insights have resulted in the identification of the noncanonical IκB kinase kinases IκB kinase ɛ and TBK1, which are upregulated by overnutrition, and may therefore be suitable potential therapeutic targets for metabolic syndromes. An inhibitor interfering with the activity of both kinases reduces obesity-related metabolic dysfunctions in mouse models and the encouraging results from a recent clinical trial indicate that targeting these NF-κB pathway components improves glucose homeostasis in a subset of patients with type 2 diabetes.


Assuntos
Metabolismo Energético , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Proteínas de Transporte/metabolismo , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/terapia , Gerenciamento Clínico , Suscetibilidade a Doenças , Glucagon/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Ligação Proteica , Serina-Treonina Quinases TOR/metabolismo
7.
J Virol ; 93(13)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30996098

RESUMO

Influenza A viruses (IAVs) quickly adapt to new environments and are well known to cross species barriers. To reveal a molecular basis for these phenomena, we compared the Ser/Thr and Tyr phosphoproteomes of murine lung epithelial cells early and late after infection with mouse-adapted SC35M virus or its nonadapted SC35 counterpart. With this analysis we identified a large set of upregulated Ser/Thr phosphorylations common to both viral genotypes, while Tyr phosphorylations showed little overlap. Most of the proteins undergoing massive changes of phosphorylation in response to both viruses regulate chromatin structure, RNA metabolism, and cell adhesion, including a focal adhesion kinase (FAK)-regulated network mediating the regulation of actin dynamics. IAV also affected phosphorylation of activation loops of 37 protein kinases, including FAK and several phosphatases, many of which were not previously implicated in influenza virus infection. Inhibition of FAK proved its contribution to IAV infection. Novel phosphorylation sites were found on IAV-encoded proteins, and the functional analysis of selected phosphorylation sites showed that they either support (NA Ser178) or inhibit (PB1 Thr223) virus propagation. Together, these data allow novel insights into IAV-triggered regulatory phosphorylation circuits and signaling networks.IMPORTANCE Infection with IAVs leads to the induction of complex signaling cascades, which apparently serve two opposing functions. On the one hand, the virus highjacks cellular signaling cascades in order to support its propagation; on the other hand, the host cell triggers antiviral signaling networks. Here we focused on IAV-triggered phosphorylation events in a systematic fashion by deep sequencing of the phosphoproteomes. This study revealed a plethora of newly phosphorylated proteins. We also identified 37 protein kinases and a range of phosphatases that are activated or inactivated following IAV infection. Moreover, we identified new phosphorylation sites on IAV-encoded proteins. Some of these phosphorylations support the enzymatic function of viral components, while other phosphorylations are inhibitory, as exemplified by PB1 Thr223 modification. Our global characterization of IAV-triggered patterns of phospho-proteins provides a rich resource to further understand host responses to infection at the level of phosphorylation-dependent signaling networks.


Assuntos
Antivirais/farmacologia , Vírus da Influenza A/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Proteoma/análise , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Genoma , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Vírus da Influenza A/genética , Camundongos , Modelos Moleculares , Fosforilação , Conformação Proteica , Proteínas Virais/química , Proteínas Virais/metabolismo
8.
FASEB J ; 33(3): 4188-4202, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30526044

RESUMO

Increasing evidence shows that many transcription factors execute important biologic functions independent from their DNA-binding capacity. The NF-κB p65 (RELA) subunit is a central regulator of innate immunity. Here, we investigated the relative functional contribution of p65 DNA-binding and dimerization in p65-deficient human and murine cells reconstituted with single amino acid mutants preventing either DNA-binding (p65 E/I) or dimerization (p65 FL/DD). DNA-binding of p65 was required for RelB-dependent stabilization of the NF-κB p100 protein. The antiapoptotic function of p65 and expression of the majority of TNF-α-induced genes were dependent on p65's ability to bind DNA and to dimerize. Chromatin immunoprecipitation with massively parallel DNA sequencing experiments revealed that impaired DNA-binding and dimerization strongly diminish the chromatin association of p65. However, there were also p65-independent TNF-α-inducible genes and a subgroup of p65 binding sites still allowed some residual chromatin association of the mutants. These sites were enriched in activator protein 1 (AP-1) binding motifs and showed increased chromatin accessibility and basal transcription. This suggests a mechanism of assisted p65 chromatin association that can be in part facilitated by chromatin priming and cooperativity with other transcription factors such as AP-1.-Riedlinger, T., Liefke, R., Meier-Soelch, J., Jurida, L., Nist, A., Stiewe, T., Kracht, M., Schmitz, M. L. NF-κB p65 dimerization and DNA-binding is important for inflammatory gene expression.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Expressão Gênica/genética , Inflamação/genética , Fator de Transcrição RelA/genética , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Dimerização , Células HeLa , Humanos , Camundongos , Ligação Proteica/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição RelB/genética
9.
PLoS Pathog ; 13(3): e1006286, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28355270

RESUMO

Coronavirus replication takes place in the host cell cytoplasm and triggers inflammatory gene expression by poorly characterized mechanisms. To obtain more insight into the signals and molecular events that coordinate global host responses in the nucleus of coronavirus-infected cells, first, transcriptome dynamics was studied in human coronavirus 229E (HCoV-229E)-infected A549 and HuH7 cells, respectively, revealing a core signature of upregulated genes in these cells. Compared to treatment with the prototypical inflammatory cytokine interleukin(IL)-1, HCoV-229E replication was found to attenuate the inducible activity of the transcription factor (TF) NF-κB and to restrict the nuclear concentration of NF-κB subunits by (i) an unusual mechanism involving partial degradation of IKKß, NEMO and IκBα and (ii) upregulation of TNFAIP3 (A20), although constitutive IKK activity and basal TNFAIP3 expression levels were shown to be required for efficient virus replication. Second, we characterized actively transcribed genomic regions and enhancers in HCoV-229E-infected cells and systematically correlated the genome-wide gene expression changes with the recruitment of Ser5-phosphorylated RNA polymerase II and prototypical histone modifications (H3K9ac, H3K36ac, H4K5ac, H3K27ac, H3K4me1). The data revealed that, in HCoV-infected (but not IL-1-treated) cells, an extensive set of genes was activated without inducible p65 NF-κB being recruited. Furthermore, both HCoV-229E replication and IL-1 were shown to upregulate a small set of genes encoding immunomodulatory factors that bind p65 at promoters and require IKKß activity and p65 for expression. Also, HCoV-229E and IL-1 activated a common set of 440 p65-bound enhancers that differed from another 992 HCoV-229E-specific enhancer regions by distinct TF-binding motif combinations. Taken together, the study shows that cytoplasmic RNA viruses fine-tune NF-κB signaling at multiple levels and profoundly reprogram the host cellular chromatin landscape, thereby orchestrating the timely coordinated expression of genes involved in multiple signaling, immunoregulatory and metabolic processes.


Assuntos
Cromatina/fisiologia , Coronavirus Humano 229E , Infecções por Coronavirus/genética , NF-kappa B/metabolismo , Transcriptoma , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Humanos , Immunoblotting , Microdissecção e Captura a Laser , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase
11.
Dev Biol ; 423(2): 170-188, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-27914912

RESUMO

Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary. We compared genome-wide expression profiles of undifferentiated E14.5 vs. abundantly ciliated E18.5 micro-dissected airway epithelia as well as Foxj1+ vs. Foxj1-deficient foetal (E16.5) lungs of the mouse using microarray hybridisation. 326 genes deregulated in both screens are candidates for FOXJ1-dependent, ciliogenesis-associated factors at the endogenous onset of motile ciliogenesis in the lung, including 123 genes that have not been linked to ciliogenesis before; 46% of these novel factors lack known homologues outside mammals. Microarray screening of Noto+ vs. Noto null early headfold embryos (E7.75) identified 59 of the lung candidates as NOTO/FOXJ1-dependent factors in the embryonic left-right organiser that carries a different subtype of motile cilia. For several uncharacterised factors from this small overlap - including 1700012B09Rik, 1700026L06Rik and Fam183b - we provide extended experimental evidence for a ciliary function.


Assuntos
Cílios/metabolismo , Feto/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Organizadores Embrionários/metabolismo , Organogênese , Mucosa Respiratória/embriologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Estudos de Associação Genética , Genoma , Proteínas de Fluorescência Verde/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Camundongos , Especificidade de Órgãos/genética , Organogênese/genética , Reprodutibilidade dos Testes , Mucosa Respiratória/citologia , Frações Subcelulares/metabolismo , Transcriptoma/genética
12.
Cell Mol Life Sci ; 74(7): 1319-1333, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27853834

RESUMO

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi's sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Antígeno CD146/genética , Antígeno CD146/metabolismo , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Endoglina/genética , Endoglina/metabolismo , Células Endoteliais/transplante , Perfilação da Expressão Gênica , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Óxido Nítrico/metabolismo , Sarcoma de Kaposi/etiologia , Regulação para Cima
13.
J Virol ; 90(17): 7980-90, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27356900

RESUMO

UNLABELLED: The role of NF-κB in influenza A virus (IAV) infection does not reveal a coherent picture, as pro- and also antiviral functions of this transcription factor have been described. To address this issue, we used clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9)-mediated genome engineering to generate murine MLE-15 cells lacking two essential components of the NF-κB pathway. Cells devoid of either the central NF-κB essential modulator (NEMO) scaffold protein and thus defective in IκB kinase (IKK) activation or cells not expressing the NF-κB DNA-binding and transactivation subunit p65 were tested for propagation of the SC35 virus, which has an avian host range, and its mouse-adapted variant, SC35M. While NF-κB was not relevant for replication of SC35M, the absence of NF-κB activity increased replication of the nonadapted SC35 virus. This antiviral effect of NF-κB was most prominent upon infection of cells with low virus titers as they usually occur during the initiation phase of IAV infection. The defect in NF-κB signaling resulted in diminished IAV-triggered phosphorylation of interferon regulatory factor 3 (IRF3) and expression of the antiviral beta interferon (IFN-ß) gene. To identify the viral proteins responsible for NF-κB dependency, reassortant viruses were generated by reverse genetics. SC35 viruses containing the SC35M segment encoding neuraminidase (NA) were completely inert to the inhibitory effect of NF-κB, emphasizing the importance of the viral genotype for susceptibility to the antiviral functions of NF-κB. IMPORTANCE: This study addresses two different issues. First, we investigated the role of the host cell transcription factor NF-κB in IAV replication by genetic manipulation of IAVs by reverse genetics combined with targeted genome engineering of host cells using CRISPR-Cas9. The analysis of these two highly defined genetic systems indicated that the IAV genotype can influence whether NF-κB displays an antiviral function and thus might in part explain incoherent results from the literature. Second, we found that perturbation of NF-κB function greatly improved the growth of a nonadapted IAV, suggesting that NF-κB may contribute to the maintenance of the host species barrier.


Assuntos
Genótipo , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Subunidade p50 de NF-kappa B/metabolismo , Replicação Viral , Animais , Linhagem Celular , Vírus da Influenza A/genética , Camundongos , Subunidade p50 de NF-kappa B/genética , Genética Reversa
15.
Biochim Biophys Acta ; 1849(8): 966-78, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26066982

RESUMO

Interleukin (IL)-36α - one of the novel members of the IL-1 family of cytokines - is a potent regulator of dendritic and T cells and plays an important role in inflammatory processes like experimental skin inflammation in mice and in mouse models for human psoriasis. Here, we demonstrate that C/EBPß, a transcription factor required for the selective expression of inflammatory genes, is a key activator of the Il36A gene in murine macrophages. RNAi-mediated suppression of C/EBPß expression in macrophages (C/EBPß(low) cells) significantly impaired Il36A gene induction following challenge with LPS. Despite the presence of five predicted C/EBP binding sites, luciferase reporter assays demonstrated that C/EBPß confers responsiveness to LPS primarily through a half-CRE•C/EBP element in the proximal Il36A promoter. Electrophoretic mobility shift assays showed that C/EBPß but not CREB proteins interact with this critical half-CRE•C/EBP element. In addition, overexpression of C/EBPß in C/EBPß(low) cells enhanced the expression of Il36A whereas CREB-1 had no effect. Finally, chromatin immunoprecipitation confirmed that C/EBPß but neither CREB-1, ATF-2 nor ATF4 is directly recruited to the proximal promoter region of the Il36A gene. Together, these findings demonstrate an essential role of C/EBPß in the regulation of the Il36A gene via the proximal half-CRE•C/EBP element in response to inflammatory stimuli.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Inflamação/genética , Interleucina-1/genética , Macrófagos/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Células Cultivadas , Códon de Iniciação/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Psoríase/genética , Elementos Reguladores de Transcrição
16.
J Immunol ; 192(7): 3143-55, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24574500

RESUMO

The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we observed blockade of IκBα phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IκBα levels were found, accompanied by increased IκBα degradation, suggesting an increased IκBα turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser(536), p38, and c-jun was inhibited, and basal inhibitory p65-Ser(468) phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and protein phosphatase 1-dependent mechanisms. To our knowledge, we present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-κB/AP-1-associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inflammation and may be involved in immune paralysis.


Assuntos
Monócitos/imunologia , NF-kappa B/imunologia , Proteína Fosfatase 1/imunologia , Transdução de Sinais/imunologia , Fator de Transcrição AP-1/imunologia , Fator de Necrose Tumoral alfa/imunologia , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Tolerância a Medicamentos/imunologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/imunologia , Quinase 3 da Glicogênio Sintase/metabolismo , Células HeLa , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/farmacologia
17.
Biochim Biophys Acta ; 1843(11): 2754-2764, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25116307

RESUMO

RNA viruses have rapidly evolving genomes which often allow cross-species transmission and frequently generate new virus variants with altered pathogenic properties. Therefore infections by RNA viruses are a major threat to human health. The infected host cell detects trace amounts of viral RNA and the last years have revealed common principles in the biochemical mechanisms leading to signal amplification that is required for mounting of a powerful antiviral response. Components of the RNA sensing and signaling machinery such as RIG-I-like proteins, MAVS and the inflammasome inducibly form large oligomers or even fibers that exhibit hallmarks of prions. Following a nucleation event triggered by detection of viral RNA, these energetically favorable and irreversible polymerization events trigger signaling cascades leading to the induction of antiviral and inflammatory responses, mediated by interferon and NF-κB pathways. Viruses have evolved sophisticated strategies to manipulate these host cell signaling pathways in order to ensure their replication. We will discuss at the examples of influenza and HTLV-1 viruses how a fascinating diversity of biochemical mechanisms is employed by viral proteins to control the NF-κB pathway at all levels.

18.
Gastroenterology ; 147(1): 184-195.e3, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24727021

RESUMO

BACKGROUND & AIMS: Activation of the transcription factor nuclear factor-κB (NF-κB) has been associated with the development of inflammatory bowel disease (IBD). Copper metabolism MURR1 domain containing 1 (COMMD1), a regulator of various transport pathways, has been shown to limit NF-κB activation. We investigated the roles of COMMD1 in the pathogenesis of colitis in mice and IBD in human beings. METHODS: We created mice with a specific disruption of Commd1 in myeloid cells (Mye-knockout [K/O] mice); we analyzed immune cell populations and functions and expression of genes regulated by NF-κB. Sepsis was induced in Mye-K/O and wild-type mice by cecal ligation and puncture or intraperitoneal injection of lipopolysaccharide (LPS), colitis was induced by administration of dextran sodium sulfate, and colitis-associated cancer was induced by administration of dextran sodium sulfate and azoxymethane. We measured levels of COMMD1 messenger RNA in colon biopsy specimens from 29 patients with IBD and 16 patients without (controls), and validated findings in an independent cohort (17 patients with IBD and 22 controls). We searched for polymorphisms in or near COMMD1 that were associated with IBD using data from the International IBD Genetics Consortium and performed quantitative trait locus analysis. RESULTS: In comparing gene expression patterns between myeloid cells from Mye-K/O and wild-type mice, we found that COMMD1 represses expression of genes induced by LPS. Mye-K/O mice had more intense inflammatory responses to LPS and developed more severe sepsis and colitis, with greater mortality. More Mye-K/O mice with colitis developed colon dysplasia and tumors than wild-type mice. We observed a reduced expression of COMMD1 in colon biopsy specimens and circulating leukocytes from patients with IBD. We associated single-nucleotide variants near COMMD1 with reduced expression of the gene and linked them with increased risk for ulcerative colitis. CONCLUSIONS: Expression of COMMD1 by myeloid cells has anti-inflammatory effects. Reduced expression or function of COMMD1 could be involved in the pathogenesis of IBD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Colite/prevenção & controle , Colite/fisiopatologia , Neoplasias do Colo/prevenção & controle , Neoplasias do Colo/fisiopatologia , Inflamação/genética , Inflamação/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Azoximetano/efeitos adversos , Biópsia , Estudos de Casos e Controles , Colite/induzido quimicamente , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/metabolismo
19.
PLoS Pathog ; 9(11): e1003737, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244164

RESUMO

Kaposi's sarcoma (KS) is a mesenchymal tumour, which is caused by Kaposi's sarcoma herpesvirus (KSHV) and develops under inflammatory conditions. KSHV-infected endothelial spindle cells, the neoplastic cells in KS, show increased invasiveness, attributed to the elevated expression of metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). The majority of these spindle cells harbour latent KSHV genomes, while a minority undergoes lytic reactivation with subsequent production of new virions and viral or cellular chemo- and cytokines, which may promote tumour invasion and dissemination. In order to better understand KSHV pathogenesis, we investigated cellular mechanisms underlying the lytic reactivation of KSHV. Using a combination of small molecule library screening and siRNA silencing we found a STE20 kinase family member, MAP4K4, to be involved in KSHV reactivation from latency and to contribute to the invasive phenotype of KSHV-infected endothelial cells by regulating COX-2, MMP-7, and MMP-13 expression. This kinase is also highly expressed in KS spindle cells in vivo. These findings suggest that MAP4K4, a known mediator of inflammation, is involved in KS aetiology by regulating KSHV lytic reactivation, expression of MMPs and COX-2, and, thereby modulating invasiveness of KSHV-infected endothelial cells.


Assuntos
Células Endoteliais/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas de Neoplasias/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Sarcoma de Kaposi/metabolismo , Ativação Viral/fisiologia , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Células Endoteliais/patologia , Células Endoteliais/virologia , Feminino , Células HEK293 , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/virologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Metaloproteinase 13 da Matriz/biossíntese , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 7 da Matriz/biossíntese , Metaloproteinase 7 da Matriz/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patologia
20.
Biochem J ; 457(3): 401-13, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24175631

RESUMO

The transcription factor NF-κB (nuclear factor κB) serves to up-regulate gene expression in response to precarious signals such as the pro-inflammatory cytokines TNF (tumour necrosis factor) and IL-1 (interleukin 1). In the present study we show that stimulation of cells with TNF or IL-1 results in a profound conformational switch of the NF-κB subunit p65, as revealed by limited proteolysis assays. We also describe the identification of a conformation-specific monoclonal antibody that preferentially immunoprecipitates the inducibly refolded p65 protein. The cytokine-triggered reconfiguration of p65 mainly occurs for p65 contained in the nuclear fraction. Phosphorylations serve as the central driving force for the inducible reconfiguration of p65. Accordingly, mutation of single phosphorylation sites in the C-terminal transactivation domain led to large conformational changes which result in strongly decreased ubiquitination and also in differential protein-protein interactions. Induced conformational changes of p65 thus increase the intramolecular flexibility and therefore expand and specify the repertoire of possible protein-protein interactions. Constitutively bound chaperones of the Hsp (heat-shock protein)/Hsc70 (heat-shock cognate protein, 73 kDa) family are not important for the cytokine-induced conformational switch, but rather control the fidelity of protein rearrangement. Accordingly, pharmacological inhibition of Hsp/Hsc70 interferes with p65-triggered gene expression.


Assuntos
Núcleo Celular/metabolismo , Citocinas/metabolismo , Processamento de Proteína Pós-Traducional , Redobramento de Proteína , Fator de Transcrição RelA/metabolismo , Regulação para Cima , Substituição de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Proteínas de Choque Térmico HSC70/antagonistas & inibidores , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fator de Transcrição RelA/química , Fator de Transcrição RelA/genética , Ubiquitinação
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