RESUMEN
Hemophilia is the deficiency of plasma clotting factor VIII (hemophilia A) or IX (hemophilia B) where management focuses on the prevention and treatment of acute bleeding symptoms and their sequelae. The most concerning risk is for life-threatening bleeding, including intracranial hemorrhage (ICH), which is caused by head trauma. Guidelines exist for the evaluation and management of pediatric head trauma, including the Pediatric Emergency Care Applied Research Network (PECARN) protocol, but limited evidence exists for when hemophilia patients present to the emergency department (ED), specifically with head trauma. Literature is limited regarding ICH and hemophilia, which further supports the culture of uncertainty among providers. The objective of this study is to conduct a retrospective chart review to determine the prevalence and clinical characteristics of ICH, and to describe computed tomography (CT) scan use in hemophilia patients who present to Phoenix Children's Hospital (PCH) ED with head trauma from January 1, 2007 to June 1, 2019. A total of 89 ED visits and 43 patients met inclusion criteria, and prevalence of ICH was determined to be 4% with the patients presenting with varied clinical characteristics and few commonalities. Using these data, we propose a new algorithm to aid clinicians in determining the need for CT scan in pediatric hemophilia patients who present to the ED with head trauma.
Asunto(s)
Traumatismos Craneocerebrales , Hemofilia A , Algoritmos , Niño , Traumatismos Craneocerebrales/complicaciones , Traumatismos Craneocerebrales/terapia , Servicio de Urgencia en Hospital , Hemofilia A/complicaciones , Hemofilia A/diagnóstico , Hemofilia A/terapia , Hemorragia/complicaciones , Humanos , Hemorragias Intracraneales/diagnóstico , Hemorragias Intracraneales/etiología , Hemorragias Intracraneales/terapia , Estudios RetrospectivosRESUMEN
Hematopoiesis and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their downstream transcription factors. A member of the mRNA export complex, THOC5 (suppressors of the transcriptional defects of hpr1 delta by overexpression complex 5) is a substrate for several tyrosine kinases such as macrophage colony-stimulating factor (M-CSF) receptor and various leukemogenic tyrosine kinases, such as Bcr-Abl, or NPM-ALK. THOC5 tyrosine phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia, suggesting that THOC5 may be involved in leukemia development. THOC5 is also an essential element in the maintenance of hematopoiesis in adult mice. In this report, we show that THOC5 is located in the nuclear speckles, and that it is translocated from the nucleus to cytoplasm during M-CSF-induced bone marrow-derived macrophage differentiation. Furthermore, we have identified THOC5 target genes by trancriptome analysis, using tamoxifen-inducible THOC5 knockout macrophages. Although only 99 genes were downregulated in THOC5-depleted macrophages, half of the genes are involved in differentiation and/or migration. These include well-known regulators of myeloid differentiation inhibitor of DNA binding (Id)1, Id3, Smad family member 6 (Smad6) and Homeobox (Hox)A1. In addition, a subset of M-CSF-inducible genes, such as Ets family mRNAs are THOC5 target mRNAs. Upon depletion of THOC5, unspliced v-ets erythroblastosis virus E26 oncogene homolog (Ets1) mRNA was accumulated in the nucleus. Furthermore, THOC5 was recruited to chromatin where Ets1 was transcribed and bound to unspliced and spliced Ets1 transcripts, indicating that THOC5 has a role in processing/export of M-CSF-inducible genes. In conclusion, regulation of immediate-early gene response by THOC5, a member of mRNA export complex contributes to the M-CSF-induced macrophage differentiation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Proteínas Nucleares/metabolismo , Transporte de ARN/genética , Transporte Activo de Núcleo Celular/efectos de los fármacos , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Células de la Médula Ósea/citología , Diferenciación Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Modelos Biológicos , Regiones Promotoras Genéticas/genética , Transporte de Proteínas/efectos de los fármacos , Proteína Proto-Oncogénica c-ets-1/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Transporte de ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
BACKGROUND AND PURPOSE: Recent studies suggested a role for PGE(2) in the expression of the chemokine IL-8. PGE(2) signals via four different GPCRs, EP(1) -EP(4) . The role of EP(1) and EP(4) receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP(1) (HEK-EP(1) ), EP(4) (HEK-EP(4) ) or both receptors (HEK-EP(1) + EP(4) ). EXPERIMENTAL APPROACH: IL-8 mRNA and protein induction and IL-8 promoter and NF-κB activation were assessed in EP expressing HEK cells. KEY RESULTS: In HEK-EP(1) and HEK-EP(1) + EP(4) but not HEK or HEK-EP(4) cells, PGE(2) activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP(1) + EP(4) cells with an EP(1) -specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP(4) agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP(1) + EP(4) cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE(2) . In HEK-EP(1) + EP(4) cells, PGE(2) -mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of IκB kinase. PGE(2) activated NF-κB in HEK-EP(1) , HEK-EP(4) and HEK-EP(1) + EP(4) cells. In HEK-EP(1) + EP(4) cells, simultaneous activation of both receptors was needed for maximal PGE(2) -induced NF-κB activation. PGE(2) -stimulated NF-κB activation by EP(1) was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP(4) was only blunted by Src-kinase inhibition. CONCLUSIONS AND IMPLICATIONS: These findings suggest that PGE(2) -mediated NF-κB activation by simultaneous stimulation of EP(1) and EP(4) receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction.
Asunto(s)
Dinoprostona/farmacología , Interleucina-8/metabolismo , FN-kappa B/metabolismo , Subtipo EP1 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Células HEK293 , Humanos , Interleucina-8/genética , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Subtipo EP1 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/genética , Transducción de SeñalRESUMEN
The nuclear factor (NF)-κB transcription factor has essential roles in inflammation and oncogenesis. Its ubiquitous RelA subunit is regulated by several post-translational modifications, including phosphorylation, ubiquitination and acetylation. Ubiquitination promotes the termination of RelA-dependent transcription, but its regulation is incompletely understood. Through mass spectrometry analysis of ubiquitinated RelA, we identified seven lysines that were attached to degradative and non-degradative forms of polyubiquitin. Interestingly, lysines targeted for acetylation were among the residues identified as ubiquitin acceptor sites. Mutation of these particular sites resulted in decreased polyubiquitination. Acetylation and ubiquitination were found to inhibit each other, consistent with their use of overlapping sites. Reconstitution of rela(-/-) fibroblasts with wild-type and mutant forms of RelA revealed that modifications at these residues can have activating and inhibitory functions depending on the target gene context. Altogether, this study elucidates that ubiquitination and acetylation can modulate each other and regulate nuclear NF-κB function in a gene-specific manner.
Asunto(s)
FN-kappa B/metabolismo , Factor de Transcripción ReIA/metabolismo , Ubiquitina/metabolismo , Acetilación , Animales , Sitios de Unión/genética , Línea Celular Tumoral , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Immunoblotting , Molécula 1 de Adhesión Intercelular/genética , Lisina/genética , Lisina/metabolismo , Ratones , Ratones Noqueados , Mutación , FN-kappa B/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción ReIA/genética , UbiquitinaciónRESUMEN
BACKGROUND: Patients with atopic dermatitis (AD) are frequently colonized with Staphylococcus aureus (S. aureus), one-third of them producing α-toxin, which is correlated with the severity of eczema in AD. Staphylococcus aureus colonizes in patients with psoriasis as well. Distinct expression of chemokine (C-C motif) ligand (CCL) and chemokine (C-X-C motif) ligand (CXCL) chemokines has been documented in both diseases. In this study, we investigated the effects of sublytic α-toxin concentrations on human macrophages that accumulate in the skin of patients with AD and psoriasis. METHODS: IFN-γ-induced protein of 10-kDa (IP-10)/CXCL10 and macrophage-derived chemokine (MDC)/CCL22 production were evaluated at the mRNA or at the protein level using qRT-PCR or ELISA, respectively. Cell surface markers' expression and chemotaxis were determined by flow cytometry and Boyden chamber technique, respectively. RESULTS: Sublytic concentrations of α-toxin strongly induced CXCL10 in macrophages at both the mRNA and the protein levels and significantly up-regulated MHC class II expression. Supernatants of α-toxin-stimulated macrophages induced the migration of human CD4+ lymphocytes via the CXCL10 receptor (CXCR3). Macrophages from patients with AD produced lower levels of CXCL10 compared to cells from patients with psoriasis as well as healthy controls in response to α-toxin. α-Toxin did not lead to a large variation in CCL22 production in macrophages from all three groups. CONCLUSIONS: Staphylococcal α-toxin contributes to Th1 polarization by induction of CXCL10 in macrophages. Macrophages from patients with AD and psoriasis responded to α-toxin in the induction of Th1-related chemokine CXCL10 diversely, which could favour the recruitment of distinct leucocyte subsets into the skin.
Asunto(s)
Toxinas Bacterianas/inmunología , Quimiocina CXCL10/biosíntesis , Dermatitis Atópica/inmunología , Macrófagos/inmunología , Staphylococcus aureus/inmunología , Western Blotting , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Separación Celular , Células Cultivadas , Quimiocina CXCL10/inmunología , Dermatitis Atópica/complicaciones , Dermatitis Atópica/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Macrófagos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones Cutáneas Estafilocócicas/complicaciones , Infecciones Cutáneas Estafilocócicas/inmunología , Células TH1/citología , Células TH1/inmunologíaRESUMEN
Constitutively active PI3K catalytic subunit alpha (PIK3CA) interfered with apoptosis induction downstream of death receptor-signaling complex formation allowing robust caspase-8 activation without triggering the execution steps of apoptosis. In mutant PIK3CA-expressing cells, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95L stimulated nuclear factor kappaB (NFkappaB) activation, invasion, and transition to an amoeboid-like morphology. NFkappaB activation and adoption of amoeboid shape were inhibited by caspase-8 knockdown or FLIP-S expression, but only the cell morphology alterations required caspase-8 activity. Furthermore, we identified caspase-8-mediated, caspase-3-independent cleavage of the protein kinase rho-associated, coiled-coil containing protein kinase 1 as a novel mechanism for acquiring amoeboid shape and enhanced invasiveness in response to TRAIL and CD95L. Taken together, we provide evidence that mutated PIK3CA converts the 'tumor surveillance' activity of cancer cell-expressed death receptors and caspase-8 toward tumor promotion.
Asunto(s)
Apoptosis/genética , Caspasa 8/metabolismo , Proteína Ligando Fas/farmacología , Mutación Missense/fisiología , Fosfatidilinositol 3-Quinasas/genética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Quinasas Asociadas a rho/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Caspasa 8/genética , Inhibidores de Caspasas , Forma de la Célula/efectos de los fármacos , Forma de la Célula/genética , Fosfatidilinositol 3-Quinasa Clase I , Inhibidores de Cisteína Proteinasa/farmacología , Células HCT116 , Humanos , Quinasa I-kappa B/metabolismo , Interleucina-8/metabolismo , Invasividad Neoplásica/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína X Asociada a bcl-2/genética , Receptor fas/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
MK2 and MK3 represent protein kinases downstream of p38 mitogen-activated protein kinase (MAPK). Deletion of the MK2 gene in mice resulted in an impaired inflammatory response although MK3, which displays extensive structural similarities and identical functional properties in vitro, is still present. Here, we analyze tumor necrosis factor (TNF) production and expression of p38 MAPK and tristetraprolin (TTP) in MK3-deficient mice and demonstrate that there are no significant differences with wild-type animals. We show that in vivo MK2 and MK3 are expressed and activated in parallel. However, the level of activity of MK2 is always significantly higher than that of MK3. Accordingly, we hypothesized that MK3 could have significant effects only in an MK2-free background and generated MK2/MK3 double-knockout mice. Unexpectedly, these mice are viable and show no obvious defects due to loss of compensation between MK2 and MK3. However, there is a further reduction of TNF production and expression of p38 and TTP in double-knockout mice compared to MK2-deficient mice. This finding, together with the observation that ectopically expressed MK3 can rescue MK2 deficiency similarly to MK2, indicates that both kinases share the same physiological function in vivo but are expressed to different levels.
Asunto(s)
Regulación de la Expresión Génica , MAP Quinasa Quinasa 3/fisiología , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesis , Animales , Células CHO , Cricetinae , Eliminación de Gen , Inflamación , Péptidos y Proteínas de Señalización Intracelular , MAP Quinasa Quinasa 3/genética , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Staphylococcus aureus is a well known trigger factor of atopic dermatitis (AD). Besides the superantigens, further exotoxins are produced by S. aureus and may have an influence on the eczema. OBJECTIVE: To explore the impact of staphylococcal alpha-toxin on human T cells, as those represent the majority of skin infiltrating cells in AD. METHODS: Adult patients with AD were screened for cutaneous colonization with alpha-toxin producing S. aureus. As alpha-toxin may induce necrosis, CD4(+) T cells were incubated with sublytic alpha-toxin concentrations. Proliferation and up-regulation of IFN-gamma on the mRNA and the protein level were assessed. The induction of t-bet translocation in CD4(+) T cells was detected with the Electrophoretic Mobility Shift Assay. RESULTS: Thirty-four percent of the patients were colonized with alpha-toxin producing S. aureus and alpha-toxin was detected in lesional skin of these patients by immunohistochemistry. Sublytic alpha-toxin concentrations induced a marked proliferation of isolated CD4(+) T cells. Microarray analysis indicated that alpha-toxin induced particularly high amounts of IFN-gamma transcripts. Up-regulation of IFN-gamma was confirmed both on the mRNA and the protein level. Stimulation of CD4(+) T cells with alpha-toxin resulted in DNA binding of t-bet, known as a key transcription factor involved into primary T helper type 1 (Th1) commitment. CONCLUSION: alpha-toxin is produced by S. aureus isolated from patients with AD. We show here for the first time that sublytic alpha-toxin concentrations activate T cells in the absence of antigen-presenting cells. Our results indicate that alpha-toxin is relevant for the induction of a Th1 like cytokine response. In AD, this facilitates the development of Th1 cell dominated chronic eczema.
Asunto(s)
Dermatitis Atópica/inmunología , Enfermedades Cutáneas Infecciosas/microbiología , Infecciones Estafilocócicas/inmunología , Células TH1/inmunología , Fosfolipasas de Tipo C/biosíntesis , Adulto , Apoptosis/inmunología , División Celular/inmunología , Células Cultivadas , Dermatitis Atópica/microbiología , Humanos , Inmunohistoquímica/métodos , Interferón gamma/genética , Interferón gamma/inmunología , Leucocitos Mononucleares/inmunología , Necrosis/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/análisis , ARN Mensajero/inmunología , Piel/inmunología , Piel/microbiología , Enfermedades Cutáneas Infecciosas/inmunología , Proteínas de Dominio T Box , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Fosfolipasas de Tipo C/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Influenza A virus infection of cells results in the induction of a variety of antiviral cytokines, including those that are regulated by transcription factors of the activating protein-1 (AP-1) family. Here we show that influenza virus infection induces AP-1-dependent gene expression in productively infected cells but not in cells that do not support viral replication. Among the AP-1 factors identified to bind to their cognate DNA element during viral infections of Madin-Darby canine kidney and U937 cells are those that are regulated via phosphorylation by JNKs. Accordingly, we observed that induction of AP-1-dependent gene expression correlates with a strong activation of JNK in permissive cells, which appears to be caused by viral RNA accumulation during replication. Blockade of JNK signaling at several levels of the cascade by transient expression of dominant negative kinase mutants and inhibitory proteins resulted in inhibition of virus-induced JNK activation, reduced AP-1 activity, and impaired transactivation of the IFN-beta promoter. Virus yields from transfected and infected cells in which JNK signaling was inhibited were higher compared with the levels from control cells. Therefore, we conclude that virus-induced activation of JNK and AP-1 is part of the innate antiviral response of the cell.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus de la Influenza A/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Factor de Transcripción AP-1/fisiología , Factor de Transcripción Activador 2 , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Activación Enzimática , Células HeLa , Humanos , Interferón beta/genética , MAP Quinasa Quinasa 7 , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , ARN Viral/metabolismo , Especificidad de la Especie , Factores de Transcripción/metabolismo , Células U937 , Replicación ViralRESUMEN
Activation of the transcription factor nuclear factor kappa B (NF-kappa B) requires its release from inhibitor of NF-kappa B (I kappa B) proteins in the cytoplasm. Much work has focussed on the identification of pathways regulating this cytosolic rate-limiting step of NF-kappa B activation. However, there is increasing evidence for another complex level of NF-kappa B activation, which involves modulatory phosphorylations of the DNA-binding subunits. These phosphorylations can control several functions of NF-kappa B, including DNA binding and transactivation properties, as well as interactions between the transcription factor and regulatory proteins. Although their overall impact on NF-kappa B function has yet to be determined, modifications of this factor will very probably provide a mechanism to fine tune NF-kappa B function.
Asunto(s)
Proteínas I-kappa B/metabolismo , FN-kappa B/metabolismo , Sitios de Unión , ADN/metabolismo , Fosforilación , Transducción de Señal , Activación TranscripcionalRESUMEN
Influenza A virus infection of cells results in the induction of a variety of antiviral cytokines, including those that are regulated by transcription factors of the activating protein-1 (AP-1) family. Here we show that influenza virus infection induces AP-1-dependent gene expression in productively infected cells but not in cells that do not support viral replication. Among the AP-1 factors identified to bind to their cognate DNA element during viral infections of Madin-Darby canine kidney and U937 cells are those that are regulated via phosphorylation by JNKs. Accordingly, we observed that induction of AP-1-dependent gene expression correlates with a strong activation of JNK in permissive cells, which appears to be caused by viral RNA accumulation during replication. Blockade of JNK signaling at several levels of the cascade by transient expression of dominant negative kinase mutants and inhibitory proteins resulted in inhibition of virus-induced JNK activation, reduced AP-1 activity, and impaired transactivation of the IFN-beta promoter. Virus yields from transfected and infected cells in which JNK signaling was inhibited were higher compared with the levels from control cells. Therefore, we conclude that virus-induced activation of JNK and AP-1 is part of the innate antiviral response of the cell.
Asunto(s)
Gripe Humana/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Orthomyxoviridae/fisiología , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Animales , Línea Celular , Regulación Viral de la Expresión Génica , Humanos , Gripe Humana/genética , Gripe Humana/virología , MAP Quinasa Quinasa 4 , Replicación ViralRESUMEN
Mechanisms of fulminant gene induction during an inflammatory response were investigated using expression of the chemoattractant cytokine interleukin-8 (IL-8) as a model. Recently we found that coordinate activation of NF-kappaB and c-Jun N-terminal protein kinase (JNK) is required for strong IL-8 transcription, whereas the p38 MAP kinase (MAPK) pathway stabilizes the IL-8 mRNA. It is unclear how these pathways are coupled to the receptor for IL-1, an important physiological inducer of IL-8. Expression of the MAP kinase kinase kinase (MAPKKK) TAK1 together with its coactivator TAB1 in HeLa cells activated all three pathways and was sufficient to induce IL-8 formation, NF-kappaB + JNK2-mediated transcription from a minimal IL-8 promoter, and p38 MAPK-mediated stabilization of a reporter mRNA containing IL-8-derived regulatory mRNA sequences. Expression of a kinase-inactive mutant of TAK1 largely blocked IL-1-induced transcription and mRNA stabilization, as well as formation of endogenous IL-8. Truncated TAB1, lacking the TAK1 binding domain, or a TAK1-derived peptide containing a TAK1 autoinhibitory domain were also efficient in inhibition. These data indicate that the previously described three-pathway model of IL-8 induction is operative in response to a physiological stimulus, IL-1, and that the MAPKKK TAK1 couples the IL-1 receptor to both transcriptional and RNA-targeted mechanisms mediated by the three pathways.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Regulación de la Expresión Génica/fisiología , Interleucina-8/genética , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas Quinasa Quinasa PAM/fisiología , Receptores de Interleucina-1/fisiología , Transcripción Genética/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Activación Enzimática , Globinas/genética , Globinas/metabolismo , Células HeLa , Humanos , Interleucina-8/biosíntesis , MAP Quinasa Quinasa 4 , Quinasas Quinasa Quinasa PAM/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/fisiología , Estabilidad del ARN , ARN Mensajero/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Activación Transcripcional , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Interleukin (IL)-8, a prototypic chemokine, is rapidly induced by the pro-inflammatory cytokine IL-1 but is barely detectable in noninduced cells. Although there is clear evidence that the transcription factor NF-kappaB plays a central role in inducible IL-8 transcription, very little is known about the cis-elements and trans-acting factors involved in silencing of the IL-8 promoter. By sequence comparison with the interferon-beta promoter, we found a negative regulatory element (NRE) in the IL-8 promoter overlapping partially with the NF-kappaB response element. Here we show that an NF-kappaB-repressing factor (NRF) binds to the IL-8 promoter NF-kappaB-NRE. Reduction of cellular NRF by expressing NRF antisense RNA results in spontaneous IL-8 gene expression. In contrast, IL-1-induced IL-8 secretion is strongly impaired by expressing NRF antisense RNA. Mutation of the NRE site results in loss of NRF binding and increased basal IL-8 transcription. On the other hand IL-1-induced IL-8 transcription is decreased by mutating the NRE. These data provide evidence for a dual role of the NRF in IL-8 transcription. Although in the absence of stimulation it is involved in transcriptional silencing, in IL-1-induced cells it is required for full induction of the IL-8 promoter.
Asunto(s)
Proteínas de Unión al ADN , Interleucina-1/fisiología , Interleucina-8/genética , FN-kappa B/antagonistas & inhibidores , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional , Secuencia de Bases , ADN , Células HeLa , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Unión Proteica , Proteínas Represoras/metabolismo , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/metabolismoAsunto(s)
Citocinas/genética , Regulación de la Expresión Génica/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transcripción Genética/inmunología , Animales , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/genética , Transducción de SeñalRESUMEN
Co-stimulation of murine EL-4 thymoma cells-carrying high numbers of TCR and type I IL-1 receptors (IL-1R)-with anti-CD3 antibodies and IL-1 resulted in synergistic enhancement of IL-2 synthesis. While the extracellular signal-regulated kinase (ERK) cascade was activated by both receptors, IL-1 preferentially stimulated Jun-N-terminal kinases (JNK) and p38 mitogen-activated kinase or microtubule-associated protein kinase (MAPK). Interruption of TCR- or IL-1R-stimulated ERK cascade by PD-98059, a specific inhibitor of MAP/ERK kinase (MEK), resulted in partial suppression of nuclear factor of activated T cells activation and in complete inhibition of IL-1-stimulated NFkappaB activation. Suppression of activation of both MEK and p38 MAPK resulted in significant inhibition of IL-2 gene expression. The results show that maximal activation of the IL-2 gene requires activation of at least two different protein kinase cascades, i.e. of the ERK and p38 pathways but presumably also that of JNK which converge at the level of the IL-2 promoter resulting in enhancement of its transcriptional activity.
Asunto(s)
Interleucina-2/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interleucina-1/metabolismo , Transducción de Señal , Linfocitos T/fisiología , Animales , Activación Enzimática , Regulación de la Expresión Génica , Interleucina-2/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Inactivation of enveloped viruses (VSV, SFV, and SHV-1) by surfactin lipopeptides was dependent on the hydrophobicity, i.e. the number of carbon atoms of the fatty acid, and on the charge of the peptide moiety as well as on the virus species. Surfactins with fatty acid chains of 13 carbon atoms showed very low antiviral activity in comparison to C14 and C15 isoforms. C15 surfactin monomethyl ester also inactivated SFV which was resistant to the mixture of surfactin isoforms as produced by Bacillus subtilis. In contrast, the dimethyl ester showed no virus-inactivation capacity. Disintegration of viral structures as determined by electron microscopy after inactivation of VSV and SFV was comparable to the titer reduction. The effect of the surfactin isoforms and methyl esters on erythrocyte hemolysis correlated with the virus-inactivation capacity. Surfactins with a fatty acid chain moiety of 15 carbon atoms and one negative charge showed the highest antiviral activity.
Asunto(s)
Antivirales/farmacología , Proteínas Bacterianas/farmacología , Hemólisis/efectos de los fármacos , Péptidos Cíclicos , Animales , Proteínas Bacterianas/química , Línea Celular , Cricetinae , Eritrocitos , Ésteres/química , Ésteres/farmacología , Herpesvirus Suido 1/efectos de los fármacos , Herpesvirus Suido 1/ultraestructura , Humanos , Técnicas In Vitro , Isomerismo , Lipopéptidos , Pulmón/metabolismo , Pulmón/virología , Microscopía Electrónica , Visón , Virus de los Bosques Semliki/efectos de los fármacos , Virus de los Bosques Semliki/ultraestructura , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/ultraestructura , Replicación Viral/efectos de los fármacosRESUMEN
A hallmark of inflammation is the burst-like formation of certain proteins, initiated by cellular stress and proinflammatory cytokines like interleukin 1 (IL-1) and tumor necrosis factor, stimuli which simultaneously activate different mitogen-activated protein (MAP) kinases and NF-kappaB. Cooperation of these signaling pathways to induce formation of IL-8, a prototype chemokine which causes leukocyte migration and activation, was investigated by expressing active and inactive forms of protein kinases. Constitutively active MAP kinase kinase 7 (MKK7), an activator of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway, induced IL-8 synthesis and transcription from a minimal IL-8 promoter. Furthermore, MKK7 synergized in both effects with NF-kappaB-inducing kinase (NIK). Activation of the IL-8 promoter by either of the kinases required functional NF-kappaB and AP-1 sites. While NIK and MKK7 did not affect degradation of IL-8 mRNA, an active form of MKK6, which selectively activates p38 MAP kinase, induced marked stabilization of the transcript and further increased IL-8 protein formation induced by NIK plus MKK7. Consistently, the MAP kinase kinase kinase MEKK1, which can activate NF-kappaB, SAPK/JNK, and p38 MAP kinases, most potently induced IL-8 formation. These results provide evidence that maximal IL-8 gene expression requires the coordinate action of at least three different signal transduction pathways which cooperate to induce mRNA synthesis and suppress mRNA degradation.
Asunto(s)
Citocinas/farmacología , Interleucina-8/biosíntesis , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Sitios de Unión , Quimiotaxis de Leucocito , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 7 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Quinasa de Factor Nuclear kappa BRESUMEN
Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline-controlled expression system to determine the half-lives of interleukin (IL)-6 and IL-8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal-induced stabilization was transferred to the stable beta-globin mRNA by a 161-nucleotide fragment of IL-8 mRNA which contains an AU-rich region, as well as by defined AU-rich elements (AREs) of the c-fos and GM-CSF mRNAs. Of the different MEKK1-activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF-kappaB pathways. Selective activation of the p38 MAP kinase (=SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant-negative mutant of p38 MAP kinase interfered with MEKK1 and also IL-1-induced stabilization. Furthermore, an active form of the p38 MAP kinase-activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6-induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress-induced gene expression by stabilizing mRNAs through an MK2-dependent, ARE-targeted mechanism.
Asunto(s)
Citocinas/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Secuencia de Bases , Citocinas/genética , Cartilla de ADN/genética , Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Interleucina-1/farmacología , Interleucina-6/genética , Interleucina-8/genética , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , ARN Mensajero/genética , Transducción de Señal , Transfección , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The cytokine interleukin-1 (IL-1) is a major inflammatory hormone which activates a broad range of genes during inflammation. The signaling mechanisms triggered by IL-1 include activation of several distinct protein kinase systems. The stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), is activated particularly strongly by the cytokine. In an attempt to delineate its role in activation of gene expression by IL-1, we inhibited the IL-1-induced SAPK/JNK activity by stable overexpression of either a catalytically inactive mutant of SAPKbeta (SAPKbeta(K-R)) or antisense RNA to SAPKbeta in human epidermal carcinoma cells. A detailed analysis of signal transduction in those cells showed that activation of neither NFkappaB nor p38 mitogen-activated protein kinase was affected, suggesting that we achieved specific blockade of the SAPK/JNK. In untransfected and vector-transfected KB cells, IL-1 induced a strong increase in expression of IL-6 and IL-8 mRNA, along with the synthesis of high amounts of the proteins. In two KB cell clones stably overexpressing the mutant SAPKbeta(K-R), and three clones stably overexpressing antisense RNA to SAPKbeta, expression of IL-6 and IL-8 in response to IL-1 was strongly reduced at both the mRNA and protein level. These data indicate that the SAPK/JNK pathway provides an indispensable signal for IL-1-induced expression of IL-6 and IL-8.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Regulación Neoplásica de la Expresión Génica/inmunología , Interleucina-1/farmacología , Interleucina-6/genética , Interleucina-8/genética , Células KB/inmunología , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Activación Enzimática , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1/fisiología , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos , Células KB/efectos de los fármacos , Células KB/enzimología , Cinética , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN sin Sentido/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Transfección , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Activation of jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) by interleukin-1 (IL-1) has been reported in many cells and in rabbit liver. Here we report selective activation of JNK/SAPK, without activation of p38 or p42 mitogen-activated protein kinases (MAPKs), by IL-1 in rabbit liver. We identified an IL-1 regulated JNK/SAPK activator present in rabbit liver using S Sepharose chromatography. It was purified and immunoprecipitated by two antisera to MAP kinase kinase 7 (MKK7). It was not recognised by an antibody to MKK4. We conclude that MKK7 is the activator of JNK/SAPK activated by IL-1 in liver and that JNK/SAPK is the only MAPK activated by IL-1 in liver.