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The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as well as therapeutic side effects such as fibrosis. We screened an in-house library of 178 substances derived from marine sponges, endophytic fungi, and higher plants, and determined their senolytic activities towards DNA damage-induced senescent HCT116 colon carcinoma cells. The Pan-PI3K-inhibitor wortmannin and its clinical derivative, PX-866, were identified to act as senolytics. PX-866 potently induced apoptotic cell death in senescent HCT116, MCF-7 mammary carcinoma, and A549 lung carcinoma cells, independently of whether senescence was induced by ionizing radiation or by chemotherapeutics, but not in proliferating cells. Other Pan-PI3K inhibitors, such as the FDA-approved drug BAY80-6946 (Copanlisib, Aliqopa®), also efficiently and specifically eliminated senescent cells. Interestingly, only the simultaneous inhibition of both PI3K class I alpha (with BYL-719 (Alpelisib, Piqray®)) and delta (with CAL-101 (Idelalisib, Zydelig®)) isoforms was sufficient to induce senolysis, whereas single application of these inhibitors had no effect. On the molecular level, inhibition of PI3Ks resulted in an increased proteasomal degradation of the CDK inhibitor p21WAF1/CIP1 in all tumor cell lines analyzed. This led to a timely induction of apoptosis in senescent tumor cells. Taken together, the senolytic properties of PI3K-inhibitors reveal a novel dimension of these promising compounds, which holds particular potential when employed alongside DNA damaging agents in combination tumor therapies.
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Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21 , Humanos , Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HCT116 , Complexo de Endopeptidases do Proteassoma/metabolismo , Apoptose/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Células MCF-7 , Proteólise/efeitos dos fármacos , Células A549 , Wortmanina/farmacologia , Senoterapia/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/genética , Dano ao DNA/efeitos dos fármacos , Pirimidinas , QuinazolinasRESUMO
Cellular responses leading to development, proliferation, and differentiation depend on RAF/MEK/ERK signaling, which integrates and amplifies signals from various stimuli for downstream cellular responses. C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, necessitating the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain, whereas SIRT4 predominantly requires the C-terminus for full interaction with C-RAF. Interestingly, SIRT4 specifically interacts with C-RAF in a pre-signaling inactive (serine 259-phosphorylated) state. Consistent with this finding, the expression of SIRT4 in HEK293 cells results in an up-regulation of pS259-C-RAF levels and a concomitant reduction in MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, we propose an additional extra-mitochondrial function of SIRT4 as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its metabolic tumor suppressor role of glutamate dehydrogenase and glutamate levels in mitochondria.
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Sirtuínas , Humanos , Células HEK293 , Sirtuínas/genética , Sirtuínas/metabolismo , Transdução de Sinais , Mitocôndrias/metabolismo , Quinases raf/genética , Quinases raf/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
SIRT4, together with SIRT3 and SIRT5, comprises the mitochondrially localized subgroup of sirtuins. SIRT4 regulates mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy) via its NAD+ -dependent enzymatic activities. Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl2 -induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, we observed that of the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4-eGFP decrease upon CoCl2 treatment of HEK293 cells. Co-treatment with BafA1, an inhibitor of autophagosome-lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132, prevented CoCl2 -induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl2 treatment and were more resistant to pseudohypoxia-induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild-type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress-induced protein degradation of SIRT4 occurs through two mechanisms: (a) via mitochondrial autophagy/mitophagy, and (b) as a separate process via proteasomal degradation within the cytoplasm.
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Lisina , Sirtuínas , Humanos , Células HEK293 , Proteínas Mitocondriais/metabolismo , Sirtuínas/metabolismo , Ubiquitinação , Complexo de Endopeptidases do Proteassoma/metabolismo , Estresse FisiológicoRESUMO
Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.
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Cardiomiopatia Hipertrófica , Síndrome de Noonan , Proteínas Proto-Oncogênicas c-raf , Humanos , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Mutação em Linhagem Germinativa , Miócitos Cardíacos/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/complicações , Síndrome de Noonan/metabolismo , Transdução de Sinais , Proteínas Proto-Oncogênicas c-raf/genéticaRESUMO
IL-6 family members contribute to host defense through the stimulation of acute-phase signaling, hematopoiesis, immune reactions, and regenerative processes. To investigate essential mechanisms that are linked toward a constitutively activated gp130 signaling, we generated and characterized a mouse model that reflects a constitutive and cytokine-independent activation of JAK/STAT3 signaling by Lgp130 in CD4- and CD8-positive T cells. Lgp130 is an engineered form of gp130 in which dimerization and activation are forced by a leucine zipper. T cell-specific Lgp130 activation resulted in massive phenotypical abnormalities, including splenomegaly, lymphadenopathy, and an upregulation of innate immune system components shown by hyperinflammatory signatures in several organs. Moreover, T cell-restricted expression of Lgp130 resulted in increased numbers of cytotoxic and regulatory T cells, especially in lymph nodes. Consistent with this, we found an elevated platelet production and increase in megakaryocytes in the spleen and bone marrow that are causative for an acute thrombocytosis accompanied by anemia. Due to a shortened life span of T cell-specific Lgp130 mice, we could also show that next to an overall increase in regulatory cell-cycle genes, an activation of p53 and increased expression of p21 provide evidence for a senescence-like phenotype. Together, these data suggest that T cell-restricted gp130 activation is not only involved in autoimmune processes but also in senescence-associated aging. Therefore, Lgp130 expression in T cells might be a suitable model to study inflammation and disease.
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Senilidade Prematura , Animais , Camundongos , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Hematopoese , Baço/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
This article contains raw and processed data related to research published by Vega et al. (2022). This complementary dataset provides further insight into the experimental validation of a single common carotid artery occlusion (CCAO) model upon pretreatment with pertussis toxin (PTX). We present data showing the extent of different PTX concentrations on neurological severity measured by Bederson score following CCAO. In addition, data indicate a protective effect of isoflurane on cerebral infarction and neurological deficits, as well as the consequences of PTX pretreatment on reperfusion after occlusion using time-of-flight magnetic resonance angiography. With these data, we aim to provide detailed experimental settings of this newly described model.
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Embryonic stem cell-expressed Ras (ERas) is an atypical constitutively active member of the Ras family and controls distinct signaling pathways, which are critical, for instance, for the maintenance of quiescent hepatic stellate cells (HSCs). Unlike classical Ras paralogs, ERas has a unique N-terminal extension (Nex) with as yet unknown function. In this study, we employed affinity pull-down and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and identified 76 novel binding proteins for human and rat ERas Nex peptides, localized in different subcellular compartments and involved in various cellular processes. One of the identified Nex-binding proteins is the nonmitochondrial, cytosolic arginase 1 (ARG1), a key enzyme of the urea cycle and involved in the de novo synthesis of polyamines, such as spermidine and spermine. Here, we show, for the first time, a high-affinity interaction between ERas Nex and purified ARG1 as well as their subcellular colocalization. The inhibition of ARG1 activity strikingly accelerates the activation of HSCs ex vivo, suggesting a central role of ARG1 activity in the maintenance of HSC quiescence.
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Arginase , Células Estreladas do Fígado , Proteína Oncogênica p21(ras) , Animais , Arginase/metabolismo , Cromatografia Líquida , Células-Tronco Embrionárias/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Proteína Oncogênica p21(ras)/metabolismo , Ratos , Espectrometria de Massas em TandemRESUMO
Silencing of the fragile X mental retardation 1 (FMR1) gene and consequently lack of synthesis of FMR protein (FMRP) are associated with fragile X syndrome, which is one of the most prevalent inherited intellectual disabilities, with additional roles in increased viral infection, liver disease, and reduced cancer risk. FMRP plays critical roles in chromatin dynamics, RNA binding, mRNA transport, and mRNA translation. However, the underlying molecular mechanisms, including the (sub)cellular FMRP protein networks, remain elusive. Here, we employed affinity pull-down and quantitative LC-MS/MS analyses with FMRP. We identified known and novel candidate FMRP-binding proteins as well as protein complexes. FMRP interacted with 180 proteins, 28 of which interacted with its N terminus. Interaction with the C terminus of FMRP was observed for 102 proteins, and 48 proteins interacted with both termini. This FMRP interactome comprises known FMRP-binding proteins, including the ribosomal proteins FXR1P, NUFIP2, Caprin-1, and numerous novel FMRP candidate interacting proteins that localize to different subcellular compartments, including CARF, LARP1, LEO1, NOG2, G3BP1, NONO, NPM1, SKIP, SND1, SQSTM1, and TRIM28. Our data considerably expand the protein and RNA interaction networks of FMRP, which thereby suggest that, in addition to its known functions, FMRP participates in transcription, RNA metabolism, ribonucleoprotein stress granule formation, translation, DNA damage response, chromatin dynamics, cell cycle regulation, ribosome biogenesis, miRNA biogenesis, and mitochondrial organization. Thus, FMRP seems associated with multiple cellular processes both under normal and cell stress conditions in neuronal as well as non-neuronal cell types, as exemplified by its role in the formation of stress granules.
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Proteínas de Transporte/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Mapas de Interação de Proteínas , Estresse Fisiológico , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Cromatografia Líquida/métodos , Proteína do X Frágil da Deficiência Intelectual/genética , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Ligação Proteica , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Astrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.
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Amônia/farmacologia , Astrócitos/metabolismo , Metabolismo Energético , Glutamato Desidrogenase/metabolismo , Aminação , Aminoácidos/metabolismo , Astrócitos/efeitos dos fármacos , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Hiperamonemia/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metaboloma/efeitos dos fármacos , Metabolômica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Sirtuínas/metabolismoRESUMO
The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,ß-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.
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Centrossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Mitose/genética , Sirtuínas/metabolismo , HumanosRESUMO
Microtubule-targeting agents (MTAs) are the most effective chemotherapeutics used in cancer therapy to date, but their clinical use is often hampered by the acquisition of resistance. Thereby, elucidation of the molecular signaling pathways activated by novel FDA-approved MTAs such as eribulin is important for future therapeutic applications. In contrast to several reports, we show here that regardless of the presence of caspase-3, clinically relevant concentrations of eribulin and the classical MTA paclitaxel predominantly induce caspase-independent cell death in MCF-7 breast carcinoma cells. On the molecular level, several key proteins involved in apoptosis such as p53, Plk1, caspase-2, and Bim as well as the two MAPKs ERK and JNK were activated by both compounds to a similar extent. However, none of them proved to be important for eribulin- and paclitaxel-induced cytotoxicity, as their siRNA-mediated knockdown or inactivation by small molecule inhibitors did not alter cell death rates. In contrast, knockdown of the anti-apoptotic Bcl-2 protein, which becomes heavily phosphorylated at Ser70 during MTA treatment, resulted surprisingly in a reduction of MTA-mediated cell death. This phenomenon can be most likely explained by our observation that the absence of Bcl-2 slowed down cell cycle progression resulting in fewer cells entering mitosis, thereby delaying the mitotic capability of these MTAs to induce cell death. Taken together, although eribulin and paclitaxel disturb the mitotic spindle differently, they exhibit no functional differences in downstream molecular cell death signaling in MCF-7 breast cancer cells.
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Caspase 3/metabolismo , Furanos/farmacologia , Cetonas/farmacologia , Microtúbulos/metabolismo , Paclitaxel/farmacologia , Transdução de Sinais , Antracenos/farmacologia , Butadienos/farmacologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Microtúbulos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Interleukin-6 (IL-6) is critically involved in liver regeneration after partial hepatectomy (PHX). Previous reports suggest that IL-6 trans-signaling through the soluble IL-6/IL-6R complex is involved in this process. However, the long-term contribution of IL-6 trans-signaling for liver regeneration after PHX is unknown. PHX-induced generation of the soluble IL-6R by ADAM (a disintegrin and metallo) proteases enables IL-6 trans-signaling, in which IL-6 forms an agonistic complex with the soluble IL-6 receptor (sIL-6R) to activate all cells expressing the signal-transducing receptor chain glycoprotein 130 (gp130). In contrast, without activation of ADAM proteases, IL-6 in complex with membrane-bound IL-6R and gp130 activates classic signaling. Here, we describe the generation of IL-6 trans-signaling mice, which exhibit boosted IL-6 trans-signaling and abrogated classic signaling by genetic conversion of all membrane-bound IL-6R into sIL-6R proteins phenocopying hyperactivation of ADAM-mediated shedding of IL-6R as single substrate. Importantly, although IL-6R deficient mice were strongly affected by PHX, survival and regeneration of IL-6 trans-signaling mice was indistinguishable from control mice, demonstrating that IL-6 trans-signaling fully compensates for disabled classic signaling in liver regeneration after PHX. Moreover, we monitored the long-term consequences of global IL-6 signaling inhibition versus IL-6 trans-signaling selective blockade after PHX by IL-6 monoclonal antibodies and soluble glycoprotein 130 as fragment crystallizable fusion, respectively. Both global IL-6 blockade and selective inhibition of IL-6 trans-signaling results in a strong decrease of overall survival after PHX, accompanied by decreased signal transducer and activator of transcription 3 phosphorylation and proliferation of hepatocytes. Mechanistically, IL-6 trans-signaling induces hepatocyte growth factor production by hepatic stellate cells. Conclusion: IL-6 trans-signaling, but not classic signaling, controls liver regeneration following PHX.
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Hepatectomia , Interleucina-6/fisiologia , Regeneração Hepática/fisiologia , Animais , Células Estreladas do Fígado/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-6/sangue , Receptores de Interleucina-6/fisiologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Human pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs. METHODS: bFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation. RESULTS: Our results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs. CONCLUSION: Collectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant.
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Fator 2 de Crescimento de Fibroblastos/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Diferenciação Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The albumin D-box binding protein (DBP) is a member of the PAR bZip (proline and acidic amino acid-rich basic leucine zipper) transcription factor family and functions as important regulator of circadian core and output gene expression. Gene expression of DBP itself is under the control of E-box-dependent binding by the Bmal1-Clock heterodimer and CRE-dependent binding by the cAMP responsive element binding protein (CREB). However, the signaling mechanism mediating CREB-dependent regulation of DBP expression in the peripheral clock remains elusive. In this study, we examined the role of the GPCR (G-protein-coupled receptor)/Gαi3 (Galphai3) controlled cAMP-CREB signaling pathway in the regulation of hepatic expression of core clock and clock-regulated genes, including Dbp. Analysis of circadian gene expression revealed that rhythmicity of hepatic transcript levels of the majority of core clock (including Per1) and clock-regulated genes were not affected by Gαi3 deficiency. Consistently, the period length of primary Gαi3 deficient tail fibroblasts expressing a Bmal1-Luciferase reporter was not affected. Interestingly, however, Gαi3 deficient female but not male mice showed a tendentiously increased activation of CREB (nuclear pSer133-CREB) accompanied by an advanced peak in Dbp gene expression and elevated mRNA levels of the cytochrome P450 family member Cyp3a11, a target gene of DBP. Accordingly, selective inhibition of CREB led to a strongly decreased expression of DBP and CYP3A4 (human Cyp3a11 homologue) in HepG2 liver cells. In summary, our data suggest that the Gαi3-pCREB signalling pathway functions as a regulator of sexual-dimorphic expression of DBP and its xenobiotic target enzymes Cyp3a11/CYP3A4.
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BACKGROUND/AIMS: From invertebrates to mammals, Gαi proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gαi3-deficiency in pre-hearing murine cochleae pointed to a role of Gαi3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary ("hair") bundle, a requirement for the progression of mature hearing. We found that the lack of Gαi3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear. METHODS: Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Gαi proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Gαi isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons. RESULTS: Here we report that lack of Gαi3 but not of the ubiquitously expressed Gαi2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Gαi2 or Gαi3 had no impact. In contrast, double-deficiency for Gαi2 and Gαi3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Gαi3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Gαi3 is selectively involved in generation of neural gain during auditory processing. CONCLUSION: We propose a so far unrecognized complexity of isoform-specific and overlapping Gαi protein functions particular during final differentiation processes.
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Proteínas de Transporte/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Audição/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Fatores de Transcrição Forkhead/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Células Ciliadas Auditivas Internas/citologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genéticaRESUMO
The stress-responsive mitochondrial sirtuin SIRT4 controls cellular energy metabolism in a NAD+-dependent manner and is implicated in cellular senescence and aging. Here we reveal a novel function of SIRT4 in mitochondrial morphology/quality control and regulation of mitophagy. We report that moderate overexpression of SIRT4, but not its enzymatically inactive mutant H161Y, sensitized cells to mitochondrial stress. CCCP-triggered dissipation of the mitochondrial membrane potential resulted in increased mitochondrial ROS levels and autophagic flux, but surprisingly led to increased mitochondrial mass and decreased Parkin-regulated mitophagy. The anti-respiratory effect of elevated SIRT4 was accompanied by increased levels of the inner-membrane bound long form of the GTPase OPA1 (L-OPA1) that promotes mitochondrial fusion and thereby counteracts fission and mitophagy. Consistent with this, upregulation of endogenous SIRT4 expression in fibroblast models of senescence either by transfection with miR-15b inhibitors or by ionizing radiation increased L-OPA1 levels and mitochondrial fusion in a SIRT4-dependent manner. We further demonstrate that SIRT4 interacts physically with OPA1 in co-immunoprecipitation experiments. Overall, we propose that the SIRT4-OPA1 axis is causally linked to mitochondrial dysfunction and altered mitochondrial dynamics that translates into aging-associated decreased mitophagy based on an unbalanced mitochondrial fusion/fission cycle.
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Envelhecimento/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Sirtuínas/metabolismo , Envelhecimento/patologia , Células Cultivadas , Senescência Celular/fisiologia , Células HEK293 , Humanos , Mitocôndrias/patologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mammalian sirtuins are fundamental regulators of a plethora of cellular functions, including gene expression, proliferation, metabolism, and ultimatively cellular aging and organismal life-span. The mitochondrial sirtuin SIRT4 acts as metabolic tumor suppressor and is down-regulated in many cancer types. We showed that SIRT4 expression was up-regulated during replicative senescence and by different anti-proliferative and senescence inducing stressors, including UVB and ionizing radiation, due to inhibition of its negative regulator, microRNA miR-15b. In our recent studies we addressed the molecular consequences of increased SIRT4 expression for mitochondrial function and quality control. We demonstrated that SIRT4 reduces O2 consumption and decreases mitochondrial membrane potential in line with an increased generation of mitochondrial reactive oxygen species (mtROS). This led to the accumulation of dysfunctional mitochondria and a more fused mitochondrial network associated with a decreased mitophagic clearance. Mechanistically, our data indicate that SIRT4 promotes mitochondrial fusion in an enzymatically dependent manner through interaction with and stabilization of the dynamin-related GTPase L-OPA1, thereby opposing fission and mitophagy. Our findings provide novel insight in the role of SIRT4 as stress triggered factor that causes mitochondrial dysfunction and impaired mitochondrial quality control through decreased mitophagy, a major hallmark of aging.
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[This corrects the article DOI: 10.1371/journal.pone.0159310.].
RESUMO
The catalytical isoforms p110γ and p110δ of phosphatidylinositide 3-kinase γ (PI3Kγ) and PI3Kδ play an important role in the pathogenesis of asthma. Two key elements in allergic asthma are increased levels of eosinophils and IgE. Dual pharmacological inhibition of p110γ and p110δ reduces asthma-associated eosinophilic lung infiltration and ameliorates disease symptoms, whereas the absence of enzymatic activity in p110γKOδD910A mice increases IgE and basal eosinophil counts. This suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. Here, we analysed mice genetically deficient for both catalytical subunits (p110γ/δ-/-) and determined basal IgE and eosinophil levels and the immune response to ovalbumin-induced asthma. Serum concentrations of IgE, IL-5 and eosinophil numbers were significantly increased in p110γ/δ-/- mice compared to single knock-out and wildtype mice. However, p110γ/δ-/- mice were protected against OVA-induced infiltration of eosinophils, neutrophils, T and B cells into lung tissue and bronchoalveolar space. Moreover, p110γ/δ-/- mice, but not single knock-out mice, showed a reduced bronchial hyperresponsiveness. We conclude that increased levels of eosinophils and IgE in p110γ/δ-/- mice do not abolish the protective effect of p110γ/δ-deficiency against OVA-induced allergic airway inflammation.