RESUMEN
Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKß is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKß's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Músculo Esquelético , Animales , Quinasas Quinasa Quinasa PAM , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Fosforilación , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.
Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Quinasas Quinasa Quinasa PAM/metabolismo , Peptidil Transferasas/metabolismo , ARN Ribosómico 18S/metabolismo , Ribosomas/metabolismo , Estrés Fisiológico , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Activación Enzimática , Células HeLa , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/genética , Conformación Proteica , Dominios Proteicos , ARN Ribosómico 18S/genética , Homología de Secuencia , Transducción de SeñalRESUMEN
BACKGROUND: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. RESULTS: We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo. CONCLUSIONS: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato , Resistencia a Antineoplásicos , Procesamiento Postranscripcional del ARN , Tristetraprolina/metabolismo , Animales , Ciclo Celular , Células Cultivadas , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Células Hep G2 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células K562 , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Células THP-1 , Transcriptoma , Tristetraprolina/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88-/-, Trif-/-, MyD88-/-Trif-/-, MK2-/-, and Zfp36-/- mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.
Asunto(s)
Necroptosis , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Tristetraprolina/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Caspasa 8/química , Caspasa 8/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Necroptosis/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Tristetraprolina/deficiencia , Tristetraprolina/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Tristetraprolin (TTP) is an RNA-binding protein and an essential factor of posttranscriptional repression of cytokine biosynthesis in macrophages. Its activity is temporally inhibited by LPS-induced p38MAPK/MAPKAPK2/3-mediated phosphorylation, leading to a rapid increase in cytokine expression. We compared TTP expression and cytokine production in mouse bone marrow-derived macrophages of different genotypes: wild type, MAPKAP kinase 2 (MK2) deletion (MK2 knockout [KO]), MK2/3 double deletion (MK2/3 double KO [DKO]), TTP-S52A-S178A (TTPaa) knock-in, as well as combined MK2 KO/TTPaa and MK2/3 DKO/TTPaa. The comparisons reveal that MK2/3 are the only LPS-induced kinases for S52 and S178 of TTP and the role of MK2 and MK3 in the regulation of TNF biosynthesis is not restricted to phosphorylation of TTP at S52/S178 but includes independent processes, which could involve other TTP phosphorylations (such as S316) or other substrates of MK2/3 or p38MAPK Furthermore, we found differences in the dependence of various cytokines on the cooperation between MK2/3 deletion and TTP mutation ex vivo. In the cecal ligation and puncture model of systemic inflammation, a dramatic decrease of cytokine production in MK2/3 DKO, TTPaa, and DKO/TTPaa mice compared with wild-type animals is observed, thus confirming the role of the MK2/3/TTP signaling axis in cytokine production also in vivo. These findings improve our understanding of this signaling axis and could be of future relevance in the treatment of inflammation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citocinas/biosíntesis , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Ratones , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas/deficienciaRESUMEN
The p38MAPK downstream targets MAPKAP kinases (MK) 2 and 3 are critical for the regulation of the macrophage response to LPS. The extents to which these two kinases act cooperatively and distinctly in regulating LPS-induced inflammatory cytokine expression are still unclear. To address this uncertainty, whole transcriptome analyses were performed using bone marrow-derived macrophages (BMDM) generated from MK2-/- or MK2/3-/- animals and their wild-type littermates. The results suggest that in BMDM, MK2 and MK3 not only cooperatively regulate the transcript expression of signaling intermediates, including IL-10, IL-19, CXCL2 and the IL-4 receptor (IL-4R)α subunit, they also exert distinct regulatory effects on the expression of specific transcripts. Based on the differential regulation of gene expression by MK2 and MK3, at least six regulatory patterns were identified. Importantly, we confirmed our previous finding, which showed that in the absence of MK2, MK3 negatively regulates IFN-ß. Moreover, this genome-wide analysis identified the regulation of Cr1A, NOD1 and Serpina3f as similar to that of IFN-ß. In the absence of MK2, MK3 also delayed the nuclear translocation of NFκB by delaying the ubiquitination and subsequent degradation of IκBß, reflecting the substantial plasticity of the response of BMDM to LPS.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transcriptoma , Animales , Células Cultivadas , Quimiocina CXCL2 , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Pathological cardiac overload induces myocardial protein synthesis and hypertrophy, which predisposes to heart failure. To inhibit hypertrophy therapeutically, the identification of negative regulators of cardiomyocyte protein synthesis is needed. Here, we identified the tumor suppressor protein TIP30 as novel inhibitor of cardiac hypertrophy and dysfunction. Reduced TIP30 levels in mice entailed exaggerated cardiac growth during experimental pressure overload, which was associated with cardiomyocyte cellular hypertrophy, increased myocardial protein synthesis, reduced capillary density, and left ventricular dysfunction. Pharmacological inhibition of protein synthesis improved these defects. Our results are relevant for human disease, since we found diminished cardiac TIP30 levels in samples from patients suffering from end-stage heart failure or hypertrophic cardiomyopathy. Importantly, therapeutic overexpression of TIP30 in mouse hearts inhibited cardiac hypertrophy and improved left ventricular function during pressure overload and in cardiomyopathic mdx mice. Mechanistically, we identified a previously unknown anti-hypertrophic mechanism, whereby TIP30 binds the eukaryotic elongation factor 1A (eEF1A) to prevent the interaction with its essential co-factor eEF1B2 and translational elongation. Therefore, TIP30 could be a therapeutic target to counteract cardiac hypertrophy.
Asunto(s)
Acetiltransferasas/metabolismo , Cardiomegalia/fisiopatología , Extensión de la Cadena Peptídica de Translación , Factores de Transcripción/metabolismo , Animales , Modelos Animales de Enfermedad , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Ratones , Ratones Endogámicos mdx , Miocitos Cardíacos/metabolismo , Factor 1 de Elongación Peptídica/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Alternative translation is an important mechanism of post-transcriptional gene regulation leading to the expression of different protein isoforms originating from the same mRNA. Here, we describe an abundant long isoform of the stress/p38MAPK-activated protein kinase MK2. This isoform is constitutively translated from an alternative CUG translation initiation start site located in the 5' UTR of its mRNA. The RNA helicase eIF4A1 is needed to ensure translation of the long and the known short isoforms of MK2, of which the molecular properties were determined. Only the short isoform phosphorylated Hsp27 in vivo, supported migration and stress-induced immediate early gene (IEG) expression. Interaction profiling revealed short-isoform-specific binding partners that were associated with migration. In contrast, the long isoform contains at least one additional phosphorylatable serine in its unique N terminus. In sum, our data reveal a longer isoform of MK2 with distinct physiological properties.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Regulación de la Expresión Génica , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Células RAW 264.7 , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , ARN Interferente PequeñoRESUMEN
Inflammatory signaling is restricted through degradation and the translational repression of cytokine mRNAs. A key factor in this regulation is tristetraprolin (TTP), an RNA-binding protein (RBP) that recruits RNA-destabilizing factors and the translation inhibitory complex 4EHP-GIGYF1/2 to AU-rich element (ARE)-containing mRNAs. Here, we show that the RBP ZNF598 contributes to the same regulatory module in a TTP-like manner. Similar to TTP, ZNF598 harbors three proline-rich motifs that bind the GYF domain of GIGYF1. RNA sequencing experiments showed that ZNF598 is required for the regulation of known TTP targets, including IL-8 and CSF2 mRNA. Furthermore, we demonstrate that ZNF598 binds to IL-8 mRNA, but not TNF mRNA. Collectively, our findings highlight that ZNF598 functions as an RBP that buffers the level of a range of mRNAs. We propose that ZNF598 is a TTP-like factor that can contribute to the regulation of the inflammatory potential of cytokine-producing cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Inflamación/metabolismo , Procesamiento Postranscripcional del ARN , Transducción de Señal , Tristetraprolina/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular Tumoral , Citocinas/metabolismo , Humanos , Inflamación/genética , Unión Proteica , ARN Mensajero/metabolismoRESUMEN
Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIPL), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIPL may allow inflammatory macrophages to participate in pathogen control for a longer duration.
Asunto(s)
Inflamación/inmunología , Proteínas Inhibidoras de la Apoptosis/inmunología , Macrófagos/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Animales , Apoptosis , Diferenciación Celular , Células Cultivadas , Macrófagos/citología , Ratones Endogámicos C57BLRESUMEN
The expression of proteins during inflammatory and immune reactions is coordinated by post-transcriptional mechanisms. A particularly strong suppression of protein expression is exerted by a conserved translational silencing element (TSE) identified in the 3' UTR of NFKBIZ mRNA, which is among the targets of the RNA-binding proteins Roquin-1/2 and MCPIP1/Regnase-1. We present evidence that in the context of the TSE MCPIP1, so far known for its endonuclease activity toward mRNAs specified by distinct stem-loop (SL) structures, also suppresses translation. Overexpression of MCPIP1 silenced translation in a TSE-dependent manner and reduced ribosome occupancy of the mRNA. Correspondingly, MCPIP1 depletion alleviated silencing and increased polysomal association of the mRNA. Translationally silenced NFKBIZ or reporter mRNAs were mostly capped, polyadenylated and ribosome associated. Furthermore, MCPIP1 silenced also cap-independent, CrPV-IRES-dependent translation. This suggests that MCPIP1 suppresses a post-initiation step. The TSE is predicted to form five SL structures. SL4 and 5 resemble target structures reported for MCPIP1 and together were sufficient for MCPIP1 binding and mRNA destabilization. Translational silencing, however, required SL1-3 in addition. Thus the NFKBIZ TSE functions as an RNA element in which sequences adjacent to the site of interaction with MCPIP1 and dispensable for accelerated mRNA degradation extend the functional repertoire of MCPIP1 to translational silencing.
Asunto(s)
Silenciador del Gen , Proteínas I-kappa B/genética , Proteínas Nucleares/genética , Biosíntesis de Proteínas , Secuencias Reguladoras de Ácido Ribonucleico , Ribonucleasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Sitios de Unión , Células HeLa , Humanos , Extensión de la Cadena Peptídica de Translación , Dominios Proteicos , ARN Mensajero/metabolismo , Receptor EphB3 , Ribonucleasas/química , Ribosomas/metabolismo , Factores de Transcripción/químicaRESUMEN
RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3'UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3'UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Inflamación/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Supervivencia Celular , Reactivos de Enlaces Cruzados , Citocinas/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunoprecipitación , Inflamación/genética , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato , TranscriptomaRESUMEN
BACKGROUND & AIMS: The kinase p38(MAPK) and its downstream target MAPKAP kinase (MK) 2 are critical regulators of inflammatory responses towards pathogens. To date, the relevance of MK2 for regulating IL-10 expression and other cytokine responses towards cytomegalovirus (CMV) infection and the impact of this pathway on viral replication in vitro and in vivo is unknown and the subject of this study. METHODS: The effect of MK2, interferon-α receptor (IFNAR)1, tristetraprolin (TTP) and IL-10 on mouse (M)CMV virus titres, cytokine expression, signal transduction, transcript stability, liver enzymes release, immune cell recruitment and aggregation in response to MCMV infection were studied ex vivo in hepatocytes and macrophages, as well as in vivo. RESULTS: MK2 is critical for MCMV-induced production of IL-10, IFN-α2 and 4, IFN-ß, IL-6, and TNF-α but not for IFN-γ. The MCMV-induced IL-10 production requires activation of IFNAR1 and is further regulated by MK2 and TTP-dependent stabilization of IL-10 transcripts. MK2(-/-) mice are able to control acute MCMV replication, despite deregulated cytokine production. This may be related to the observation that MCMV-infected MK2(-/-) mice show enhanced formation of focal intrahepatic lymphocyte infiltrates resembling intrahepatic myeloid cell aggregates of T cell expansion (iMATEs), which were also observed in MCMV-infected IL-10(-/-) mice but are almost absent in MCMV-infected wild-type controls. CONCLUSIONS: The data suggest that MK2 is critical for regulating cytokine responses towards acute MCMV infection, including that of IL-10 via IFNARI-mediated circuits. MCMV stimulates expression of MK2-dependent cytokines, in particular IL-10 and thereby prevents enhanced formation of intrahepatic iMATE-like cellular aggregates.
Asunto(s)
Infecciones por Citomegalovirus , Interleucina-10/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado , Células Mieloides/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Agregación Celular/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/patología , Interferón-alfa/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones , Receptor de Interferón alfa y beta/metabolismo , Tristetraprolina/metabolismoRESUMEN
Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.
Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Fenantrenos/farmacología , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias de la Mama , Línea Celular Tumoral , Citoplasma/metabolismo , Resistencia a Antineoplásicos/genética , Proteína 1 Similar a ELAV/genética , Femenino , Furanos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Fenantrenos/toxicidad , Polirribosomas/metabolismo , Unión Proteica/efectos de los fármacos , Quinonas , Proteínas de Unión al ARN/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
MK5 (MAPK-activated protein kinase 5) or PRAK (p38-regulated and -activated kinase) are alternative names for a serine/threonine protein kinase downstream to ERK3/4 and p38 MAPK. A previous gene targeting approach for MK5/PRAK (termed here MK5/PRAK-Δex8) revealed a seemingly tumor-suppressive role of MK5/PRAK in DMBA-induced one step skin carcinogenesis and Ras-induced transformation. Here we demonstrate that an alternative targeting strategy of MK5/PRAK (termed MK5/PRAK-Δex6) increased neither tumor incidence in the one step skin carcinogenesis model, nor Ras-induced transformation in primary cells. Interestingly, due to the targeting strategies and exon skipping both knockouts do not completely abolish the generation of MK5/PRAK protein, but express MK5/PRAK deletion mutants with different biochemical properties depending on the exon targeted: Targeting of exon 6 leads to expression of an unstable cytoplasmic catalytically inactive MK5/PRAK-Δex6 mutant while targeting of exon 8 results in a more stable nuclear MK5/PRAK-Δex8 mutant with residual catalytic activity. The different properties of the MK5/PRAK deletion mutants could be responsible for the observed discrepancy between the knockout strains and challenge the role of MK5/PRAK in p53-dependent tumor suppression. Further MK5/PRAK knockout and knock-in mouse strains will be necessary to assign a physiological function to MK5/PRAK in this model organism.
Asunto(s)
Técnicas de Inactivación de Genes/métodos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Piel/patología , Proteínas Supresoras de Tumor/genética , Animales , Células Cultivadas , Exones , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Piel/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas Supresoras de Tumor/análisis , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The nuclear exosome targeting complex (NEXT) directs a major 3'-5' exonuclease, the RNA exosome, for degradation of nuclear noncoding (nc) RNAs. We identified the RNA-binding component of the NEXT complex, RBM7, as a substrate of p38(MAPK)/MK2-mediated phosphorylation at residue S136. As a result of this phosphorylation, RBM7 displays a strongly decreased RNA-binding capacity, while inhibition of p38(MAPK) or mutation of S136A in RBM7 increases its RNA association. Interestingly, promoter-upstream transcripts (PROMPTs), such as proRBM39, proEXT1, proDNAJB4, accumulated upon stress stimulation in a p38(MAPK)/MK2-dependent manner, a process inhibited by overexpression of RBM7(S136A). While there are no stress-dependent changes in RNA-polymerase II (RNAPII) occupation of PROMPT regions representing unchanged transcription, stability of PROMPTs is increased. Hence, we propose that phosphorylation of RBM7 by the p38(MAPK)/MK2 axis increases nuclear ncRNA stability by blocking their RBM7-binding and subsequent RNA exosome targeting to allow stress-dependent modulations of the noncoding transcriptome.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Exosomas , Células HEK293 , Células HeLa , Humanos , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Estabilidad del ARN , ARN no Traducido/genética , ARN no Traducido/metabolismo , Estrés FisiológicoRESUMEN
Extracellular-regulated kinases and p38 mitogen-activated protein kinases are activated in innate (and adaptive) immunity and signal via different routes to alter the stability and translation of various cytokine mRNAs, enabling immune cells to respond promptly. This regulation involves mRNA elements, such as AU-rich motifs, and mRNA-binding proteins, such as tristetraprolin (TTP), HuR, and hnRNPK-homology (KH) type splicing regulatory protein (KSRP). Signal-dependent phosphorylation of mRNA-binding proteins often alters their subcellular localization or RNA-binding affinity. Furthermore, it could lead to an altered interaction with other mRNA-binding proteins and altered scaffolding properties for mRNA-modifying enzymes, such as deadenylases, polyadenylases, decapping enzymes, poly(A) binding proteins, exo- or endonucleases, and proteins of the exosome machinery. In many cases, this results in unstable mRNAs being stabilized, with their translational arrest being released and cytokine production being stimulated. Hence, components of these mechanisms are potential targets for the modulation of the inflammatory response.
Asunto(s)
Citocinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , ARN Mensajero/metabolismo , Inmunidad Adaptativa , Animales , Citocinas/genética , Proteínas ELAV/metabolismo , Humanos , Inmunidad Innata , Mamíferos , Fosforilación , Biosíntesis de Proteínas/inmunología , Estabilidad del ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/inmunología , Transactivadores/metabolismo , Tristetraprolina/metabolismoRESUMEN
The p38 MAPK (mitogen-activated protein kinase)/MK2 [MAPKAP (MAPK-activated protein) kinase-2] signalling pathway is a major regulator of stress- and cytokine-induced gene expression at the transcriptional and post-transcriptional level. Using phosphoproteomics we identified the ER (endoplasmic reticulum)-associated ubiquitin-conjugating enzyme Ube2j1 as a potential substrate of MK2. We demonstrate that Ube2j1 is phosphorylated in a cytokine-, cytosolic stress- and LPS (lipopolysaccharide)-induced manner. The cytosolic stress-induced phosphorylation of Ube2j1 proceeds at Ser(184), a site described previously to be phosphorylated in response to ER stress, which is located in a perfect MK2 consensus motif. The cytosolic stress-induced phosphorylation of Ube2j1, but not its ER-stress-induced phosphorylation is sensitive to p38/MK2 inhibitors and abrogated in MK2/MK3-deficient cells. In a pull-down assay we demonstrate the interaction of MK2 with Ube2j1 in HEK (human embryonic kidney)-293T cells. Furthermore, MK2 is able to phosphorylate recombinant Ube2j1, but not the S184A mutant in an in vitro kinase assay. These findings strongly suggest that MK2 directly phosphorylates Ube2j1 at Ser(184) upon p38-activating stress in vivo. However, ectopically expressed Ube2j1-S184A mutant displays ubiquitinating activity towards the model substrate ER-synthesized T-cell receptor-α similar to that of the wild-type protein. Interestingly, Ube2j1 is phosphorylated in response to LPS also in macrophages and contributes to MK2-dependent TNFα biosynthesis by a so far unknown mechanism.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Animales , Línea Celular , Estrés del Retículo Endoplásmico , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteolisis , Estrés Fisiológico , Receptores Toll-Like/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The production of the proinflammatory cytokines TNF-α and IL-6 is regulated by various mRNA-binding proteins, influencing stability and translation of the respective transcripts. Research in macrophages has shown the importance of the p38-MK2-tristetraprolin (TTP) axis for regulation of TNF-α mRNA stability and translation. In the current study we examined a possible involvement of p38 and TTP in LPS-induced cytokine production in bone marrow-derived mast cells (BMMCs). Using pharmacological inhibitors we initially found a strong dependence of LPS-induced TNF-α and IL-6 production on p38 activation, whereas activation of the Erk pathway appeared dispensable. LPS treatment also induced p38-dependent expression of the TTP gene. This prompted us to analyze the proinflammatory cytokine response in BMMCs generated from TTP-deficient mice. Unexpectedly, there were no significant differences in cytokine production between TTP-deficient and WT BMMCs in response to LPS. Gene expression and cytokine production of TNF-α and IL-6 as well as stability of the TNF-α transcript were comparable between TTP-deficient and WT BMMCs. In contrast to TTP mRNA expression, TTP protein expression could not be detected in BMMCs. While we successfully precipitated and detected TTP from lysates of LPS-stimulated RAW 264.7 macrophages, this was not accomplished from BMMC lysates. In contrast, we found mRNA and protein expressions of the other TIS11 family members connected to regulation of mRNA stability, BRF1 and BRF2, and detected their interaction with 14-3-3 proteins. These data suggest that control of cytokine mRNA stability and translation in MCs is exerted by proteins different from TTP.
Asunto(s)
Interleucina-6/metabolismo , Tristetraprolina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Factor 1 de Respuesta al Butirato , Línea Celular , Interleucina-6/genética , Lipopolisacáridos/toxicidad , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Tristetraprolina/deficiencia , Tristetraprolina/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU-rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE-binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTP's own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.