RESUMO
Excessive calorie intake leads to mitochondrial overload and triggers metabolic inflexibility and insulin resistance. In this study, we examined how attenuated p38α activity affects glucose and fat metabolism in the skeletal muscles of mice on a high-fat diet (HFD). Mice exhibiting diminished p38α activity (referred to as p38αAF) gained more weight and displayed elevated serum insulin levels, as well as a compromised response in the insulin tolerance test, compared to the control mice. Additionally, their skeletal muscle tissue manifested impaired insulin signaling, leading to resistance in insulin-mediated glucose uptake. Examination of muscle metabolites in p38αAF mice revealed lower levels of glycolytic intermediates and decreased levels of acyl-carnitine metabolites, suggesting reduced glycolysis and ß-oxidation compared to the controls. Additionally, muscles of p38αAF mice exhibited severe abnormalities in their mitochondria. Analysis of myotubes derived from p38αAF mice revealed reduced mitochondrial respiratory capacity relative to the myotubes of the control mice. Furthermore, these myotubes showed decreased expression of Acetyl CoA Carboxylase 2 (ACC2), leading to increased fatty acid oxidation and diminished inhibitory phosphorylation of pyruvate dehydrogenase (PDH), which resulted in elevated mitochondrial pyruvate oxidation. The expected consequence of reduced mitochondrial respiratory function and uncontrolled nutrient oxidation observed in p38αAF myotubes mitochondrial overload and metabolic inflexibility. This scenario explains the increased likelihood of insulin resistance development in the muscles of p38αAF mice compared to the control mice on a high-fat diet. In summary, within skeletal muscles, p38α assumes a crucial role in orchestrating the mitochondrial adaptation to caloric surplus by promoting mitochondrial biogenesis and regulating the selective oxidation of nutrients, thereby preventing mitochondrial overload, metabolic inflexibility, and insulin resistance.
Assuntos
Dieta Hiperlipídica , Resistência à Insulina , Proteína Quinase 14 Ativada por Mitógeno , Músculo Esquelético , Animais , Camundongos , Músculo Esquelético/metabolismo , Dieta Hiperlipídica/efeitos adversos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/genética , Masculino , Mitocôndrias/metabolismo , Insulina/metabolismo , Insulina/sangue , Oxirredução , Adaptação Fisiológica , Glucose/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismoRESUMO
Glioblastoma (GBM) is the most aggressive and lethal brain tumor in adults. This study aimed to investigate the functional significance of aryl hydrocarbon receptor nuclear translocator (ARNT) in the pathogenesis of GBM. Analysis of public datasets revealed ARNT is upregulated in GBM tissues compared to lower grade gliomas or normal brain tissues. Higher ARNT expression correlated with the mesenchymal subtype and poorer survival in GBM patients. Silencing ARNT using lentiviral shRNAs attenuated the proliferative, invasive, and stem-like capabilities of GBM cell lines, while ARNT overexpression enhanced these malignant phenotypes. Single-cell RNA sequencing uncovered that ARNT is highly expressed in a stem-like subpopulation and is involved in regulating glycolysis, hypoxia response, and stress pathways. Mechanistic studies found ARNT activates p38 mitogen-activated protein kinase (MAPK) signaling to promote chemoresistance in GBM cells. Disrupting the ARNT/p38α protein interaction via the ARNT PAS-A domain restored temozolomide sensitivity. Overall, this study demonstrates ARNT functions as an oncogenic driver in GBM pathogenesis and represents a promising therapeutic target.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/genética , Camundongos , Regulação Neoplásica da Expressão Gênica , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Camundongos Nus , Transdução de Sinais/efeitos dos fármacosRESUMO
Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis - effects determined to be mainly dependent on MKP1's dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.
Assuntos
Fosfatase 1 de Especificidade Dupla , Pulmão , Proteína Quinase 14 Ativada por Mitógeno , Miofibroblastos , Fibrose Pulmonar , Animais , Camundongos , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatase 1 de Especificidade Dupla/genética , Miofibroblastos/patologia , Miofibroblastos/metabolismo , Miofibroblastos/enzimologia , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/induzido quimicamente , Pulmão/patologia , Pulmão/metabolismo , Bleomicina/toxicidade , Humanos , Camundongos Knockout , Camundongos Transgênicos , ApoptoseRESUMO
The small-molecule drug ralimetinib was developed as an inhibitor of the p38α mitogen-activated protein kinase, and it has advanced to phase 2 clinical trials in oncology. Here, we demonstrate that ralimetinib resembles EGFR-targeting drugs in pharmacogenomic profiling experiments and that ralimetinib inhibits EGFR kinase activity in vitro and in cellulo. While ralimetinib sensitivity is unaffected by deletion of the genes encoding p38α and p38ß, its effects are blocked by expression of the EGFR-T790M gatekeeper mutation. Finally, we solved the cocrystal structure of ralimetinib bound to EGFR, providing further evidence that this drug functions as an ATP-competitive EGFR inhibitor. We conclude that, though ralimetinib is >30-fold less potent against EGFR compared to p38α, its ability to inhibit EGFR drives its primary anticancer effects. Our results call into question the value of p38α as an anticancer target, and we describe a multi-modal approach that can be used to uncover a drug's mechanism-of-action.
Assuntos
Neoplasias Pulmonares , Proteína Quinase 14 Ativada por Mitógeno , Humanos , Receptores ErbB , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Mutação , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismoRESUMO
BACKGROUND: Thyroid cancer is one of the most prevalent malignancies worldwide. Genetic and epigenetic alterations are one of the main causes of thyroid tumor that is responsible to the activation of oncogenes as well as the inactivation of tumor suppressor genes. This research aimed to investigate the relationship of promoter methylation patterns with the expression of P38α in Iranian patients with thyroid cancer. METHODS: We collected 40 thyroid tumor samples and 40 adjacent normal thyroid samples from 40 Iranian patients with papillary thyroid cancer. The promoter methylation pattern of P38α gene was investigated by methylation-sensitive high-resolution melting (MS-HRM) method. Moreover, mRNA expression of P38α was investigated by Real-Time PCR method. Further validation of the obtained results was performed by the Cancer Genome Atlas (TCGA) dataset. RESULTS: The obtained results indicated that the expression of the P38α (MAPK-14) gene in the thyroid cancer sample was considerably higher than tumor margin sample. Also, P38α gene promoter methylation was higher in thyroid margin tissue as compared to tumor tissue. These results were additionally confirmed by TCGA analysis. The receiver operating characteristic (ROC) curve analysis showed a high accuracy of P38α gene expression as a diagnostic biomarker for thyroid malignancy. CONCLUSION: Our study demonstrated that the P38α expression level gene was associated with thyroid cancer pathogenesis among the Iranian population. We suggested that this gene expression might be used as a biomarker for diagnosis of thyroid tumor.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno , Neoplasias da Glândula Tireoide , Humanos , Câncer Papilífero da Tireoide/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Metilação de DNA , Irã (Geográfico)/epidemiologia , Neoplasias da Glândula Tireoide/patologia , Biomarcadores/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
The p38 members of the mitogen-activated protein kinases (MAPKs) family mediate various cellular responses to stress conditions, inflammatory signals, and differentiation factors. They are constitutively active in chronic inflammatory diseases and some cancers. The differences between their transient effects in response to signals and the chronic effect in diseases are not known. The family is composed of four isoforms, of which p38α seems to be abnormally activated in diseases. p38α and p38ß are almost identical in sequence, structure, and biochemical and pharmacological properties, and the specific unique effects of each of them, if any, have not yet been revealed. This study aimed to reveal the specific effects induced by p38α and p38ß, both when transiently activated in response to stress and when chronically active. This was achieved via large-scale proteomics and phosphoproteomics analyses using stable isotope labeling of two experimental systems: one, mouse embryonic fibroblasts (MEFs) deficient in each of these p38 kinases and harboring either an empty vector or vectors expressing p38αWT, p38ßWT, or intrinsically active variants of these MAPKs; second, induction of transient stress by exposure of MEFs, p38α-/-, and p38ß-/- MEFs to anisomycin. Significant differences in the repertoire of the proteome and phosphoproteome between cells expressing active p38α and p38ß suggest distinct roles for each kinase. Interestingly, in both cases, the constitutive activation induced adaptations of the cells to the chronic activity so that known substrates of p38 were downregulated. Within the dramatic effect of p38s on the proteome and phosphoproteome, some interesting affected phosphorylation sites were those found in cancer-associated p53 and Hspb1 (HSP27) proteins and in cytoskeleton-associated proteins. Among these, was the stronger direct phosphorylation by p38α of p53-Ser309, which was validated on the Ser315 in human p53. In summary, this study sheds new light on the differences between chronic and transient p38α and p38ß signaling and on the specific targets of these two kinases.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno , Proteoma , Animais , Humanos , Camundongos , Proteoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fibroblastos/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
AIMS/HYPOTHESIS: Hyperglucagonaemia-stimulated hepatic glucose production (HGP) contributes to hyperglycaemia during type 2 diabetes. A better understanding of glucagon action is important to enable efficient therapies to be developed for the treatment of diabetes. Here, we aimed to investigate the role of p38 MAPK family members in glucagon-induced HGP and determine the underlying mechanisms by which p38 MAPK regulates glucagon action. METHODS: p38α, ß, γ and δ MAPK siRNAs were transfected into primary hepatocytes, followed by measurement of glucagon-induced HGP. Adeno-associated virus serotype 8 carrying p38α MAPK short hairpin RNA (shRNA) was injected into liver-specific Foxo1 knockout, liver-specific Irs1/Irs2 double knockout and Foxo1S273D knockin mice. Foxo1S273A knockin mice were fed a high-fat diet for 10 weeks. Pyruvate tolerance tests, glucose tolerance tests, glucagon tolerance tests and insulin tolerance tests were carried out in mice, liver gene expression profiles were analysed and serum triglyceride, insulin and cholesterol levels were measured. Phosphorylation of forkhead box protein O1 (FOXO1) by p38α MAPK in vitro was analysed by LC-MS. RESULTS: We found that p38α MAPK, but not the other p38 isoforms, stimulates FOXO1-S273 phosphorylation and increases FOXO1 protein stability, promoting HGP in response to glucagon stimulation. In hepatocytes and mouse models, inhibition of p38α MAPK blocked FOXO1-S273 phosphorylation, decreased FOXO1 levels and significantly impaired glucagon- and fasting-induced HGP. However, the effect of p38α MAPK inhibition on HGP was abolished by FOXO1 deficiency or a Foxo1 point mutation at position 273 from serine to aspartic acid (Foxo1S273D) in both hepatocytes and mice. Moreover, an alanine mutation at position 273 (Foxo1S273A) decreased glucose production, improved glucose tolerance and increased insulin sensitivity in diet-induced obese mice. Finally, we found that glucagon activates p38α through exchange protein activated by cAMP 2 (EPAC2) signalling in hepatocytes. CONCLUSIONS/INTERPRETATION: This study found that p38α MAPK stimulates FOXO1-S273 phosphorylation to mediate the action of glucagon on glucose homeostasis in both health and disease. The glucagon-induced EPAC2-p38α MAPK-pFOXO1-S273 signalling pathway is a potential therapeutic target for the treatment of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2 , Proteína Quinase 14 Ativada por Mitógeno , Animais , Camundongos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Glucagon/metabolismo , Gluconeogênese/genética , Glucose/metabolismo , Hepatócitos/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , FosforilaçãoRESUMO
Since the introduction of a highly pathogenic genotype II isolate of the African swine fever virus (ASFV) into Georgia in 2007, African swine fever (ASF) has gone panzootic. Outbreaks have been reported in Europe, Asia and, more recently, Latin America. Thus, ASFV has become a major threat to the pig industry worldwide, as broadly applicable vaccines are not available. While the majority of ASFV strains show high virulence in domestic pigs and wild boar, variations within the ASFV genome have resulted in the emergence of attenuated strains with low or moderate virulence. However, the molecular basis of the differences in virulence has not yet been discovered. To reveal virulence-associated protein expression patterns, we analysed the proteomes of the natural target cells of ASFV, primary porcine macrophages, after infection with two genotype II ASFV strains displaying high (Armenia 2008) and moderate (Estonia 2014) virulence using quantitative mass spectrometry. Very similar expression patterns were observed for the viral genes, and any differences were limited to the deletions within the Estonia 2014 genome. In addition to the canonical ASFV proteins, twelve novel protein products from recently described transcripts were confirmed in both isolates. Pathway analyses showed that both isolates evoked a similar host proteome response, despite their difference in virulence. However, subtle differences in the manipulation of the proteins involved in the proinflammatory response mediated by the MAPK14/p38 signalling cascade were observed.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Proteína Quinase 14 Ativada por Mitógeno , Vacinas Virais , Suínos , Animais , Proteoma/genética , Virulência , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Macrófagos , Sus scrofa , GenótipoRESUMO
Background: Sepsis leads to multiple organ dysfunction caused by a dysregulated host response to infection with a high incidence and mortality. The effect of ferroptosis on the development of sepsis remains unclear. In this study, we aimed to identify the key ferroptosis-related genes involved in sepsis and further explore the potential biological functions of these ferroptosis-related genes in sepsis using bioinformatics analysis. Methods: The GSE13904 (from children) and GSE28750 (from adults) datasets were downloaded from the Gene Expression Omnibus (GEO). The ferroptosis-related genes were obtained from the FerrDb database. The ferroptosis-related differentially expressed genes (DEGs) were screened by the limma R package. The DAVID online database or clusterProfiler R package was used for the functional enrichment analysis. Then, the STRING database was used to predict the interactions of proteins, and the CytoHubba plugin of Cytoscape was used to confirm key clustering modules. Then, the miRNAs and lncRNAs associated with the key clustering modules were predicted by miRWalk 2.0 and LncBase v.2 respectively. Finally, we generated a cecal ligation and puncture (CLP) polymicrobial sepsis model in C57 male mice and examined the expression of the mRNAs and noncoding RNAs of interest in peripheral blood leukocytes by PCR during the acute inflammation phase. Results: In total, 34 ferroptosis-related DEGs were identified in both adult and pediatric septic patients. These ferroptosis-related DEGs were mainly enriched in inflammatory pathways. Then, a significant clustering module containing eight genes was identified. Among them, the following five genes were closely associated with the MAPK signaling pathway: MAPK14, MAPK8, DUSP1, MAP3K5 and MAPK1. Then, crucial miRNAs and lncRNAs associated with biomarker MAPK-related genes were also identified. In particular, let-7b-5p and NEAT1 were selected as noncoding RNAs of interest because of their correlation with ferroptosis in previous studies. Finally, we examined the mRNAs, miRNAs and lncRNAs of interest using CLP-induced sepsis in peripheral blood leukocytes of mice. The results showed that MAPK14, MAPK8, MAP3K5, MAPK1 and NEAT1 were upregulated, while DUSP1 and let-7b-5p were downregulated in the CLP group compared with the sham group. Conclusions: The MAPK signaling pathway may play a key role in regulating ferroptosis during sepsis. This study provides a valuable resource for future studies investigating the mechanism of MAPK-related ferroptosis in sepsis.
Assuntos
Ferroptose , MicroRNAs , Proteína Quinase 14 Ativada por Mitógeno , RNA Longo não Codificante , Sepse , Masculino , Camundongos , Animais , Perfilação da Expressão Gênica , RNA Longo não Codificante/genética , Ferroptose/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , MicroRNAs/genética , RNA Mensageiro/genética , Sepse/genéticaRESUMO
Alzheimer's disease (AD), the most common cause of dementia in the elderly, is pathologically characterized by extracellular deposition of amyloid-ß peptides (Aß) and microglia-dominated inflammatory activation in the brain. p38α-MAPK is activated in both neurons and microglia. How p38α-MAPK in microglia contributes to AD pathogenesis remains unclear. In this study, we conditionally knocked out p38α-MAPK in all myeloid cells or specifically in microglia of APP-transgenic mice, and examined animals for AD-associated pathologies (i.e., cognitive deficits, Aß pathology, and neuroinflammation) and individual microglia for their inflammatory activation and Aß internalization at different disease stages (e.g., at 4 and 9 months of age). Our experiments showed that p38α-MAPK-deficient myeloid cells were more effective than p38α-MAPK-deficient microglia in reducing cerebral Aß and neuronal impairment in APP-transgenic mice. Deficiency of p38α-MAPK in myeloid cells inhibited inflammatory activation of individual microglia at 4 months but enhanced it at 9 months. Inflammatory activation promoted microglial internalization of Aß. Interestingly, p38α-MAPK-deficient myeloid cells reduced IL-17a-expressing CD4-positive lymphocytes in 9 but not 4-month-old APP-transgenic mice. By cross-breeding APP-transgenic mice with Il-17a-knockout mice, we observed that IL-17a deficiency potentially activated microglia and reduced Aß deposition in the brain as shown in 9-month-old myeloid p38α-MAPK-deficient AD mice. Thus, p38α-MAPK deficiency in all myeloid cells, but not only in microglia, prevents AD progression. IL-17a-expressing lymphocytes may partially mediate the pathogenic role of p38α-MAPK in peripheral myeloid cells. Our study supports p38α-MAPK as a therapeutic target for AD patients.
Assuntos
Doença de Alzheimer , Proteína Quinase 14 Ativada por Mitógeno , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Interleucina-17/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia , Proteína Quinase 14 Ativada por Mitógeno/genética , Células MieloidesRESUMO
BACKGROUND: Hepatocellular Carcinoma (HCC) is recognized as the second leading cause of cancer-associated deaths globally. Hypoxia-inducible factor 1alpha (HIF1A) has been documented to promote HCC cell migration, invasion and cell cycle. Dual specificity phosphatase 18 (DUSP18) has been predicted to be up-regulated in hypoxia and its expression is positively linked to HIF1A expression in HCC cells. However, their function and molecular mechanism have not been investigated in HCC in depth. PURPOSE: This study aimed to uncover the functional roles of HIF1A and DUSP18, as well as relevant mechanisms underlying their regulation in HCC cells. METHODS: RT-qPCR and western blot were performed to examine gene expression. Functional assays were implemented to reveal the regulatory impact of target genes on HCC cells. Mechanism experiments were conducted to analyze gene interaction. RESULTS: DUSP18 was found to have significantly high expression in hypoxia-induced HCC cells. HIF1A promoted HCC cell migration, invasion and cell cycle by transcriptionally activating DUSP18. DUSP18 mediated MAPK14 dephosphorylation to weaken MAPK14 activity, which further inhibited MAPK14-mediated TP53 phosphorylation, consequently promoting multiple biological behaviors of HCC cells. CONCLUSION: Hypoxia-induced HIF1A activates DUSP18 transcription to further promote MAPK14 dephosphorylation, thereby suppressing TP53 phosphorylation and functionally promoting malignant behaviors of HCC cells.
Assuntos
Carcinoma Hepatocelular , Fosfatases de Especificidade Dupla , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias Hepáticas , Proteína Quinase 14 Ativada por Mitógeno , Humanos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Regulação Neoplásica da Expressão Gênica , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/patologia , Proteína Quinase 14 Ativada por Mitógeno/genética , Transdução de Sinais/genéticaRESUMO
Glioblastoma multiforme (GBM) is the most aggressive and prevalent form of brain cancer, with an expected survival of 12-15 months following diagnosis. GBM affects the glial cells of the central nervous system, which impairs regular brain function including memory, hearing, and vision. GBM has virtually no long-term survival even with treatment, requiring novel strategies to understand disease progression. Here, we identified a somatic mutation in OR2T7, a G-protein-coupled receptor (GPCR), that correlates with reduced progression-free survival for glioblastoma (log rank p-value = 0.05), suggesting a possible role in tumor progression. The mutation, D125V, occurred in 10% of 396 glioblastoma samples in The Cancer Genome Atlas, but not in any of the 2504 DNA sequences in the 1000 Genomes Project, suggesting that the mutation may have a deleterious functional effect. In addition, transcriptome analysis showed that the p38α mitogen-activated protein kinase (MAPK), c-Fos, c-Jun, and JunB proto-oncogenes, and putative tumor suppressors RhoB and caspase-14 were underexpressed in glioblastoma samples with the D125V mutation (false discovery rate < 0.05). Molecular modeling and molecular dynamics simulations have provided preliminary structural insight and indicate a dynamic helical movement network that is influenced by the membrane-embedded, cytofacial-facing residue 125, demonstrating a possible obstruction of G-protein binding on the cytofacial exposed region. We show that the mutation impacts the "open" GPCR conformation, potentially affecting Gα-subunit binding and associated downstream activity. Overall, our findings suggest that the Val125 mutation in OR2T7 could affect glioblastoma progression by downregulating GPCR-p38 MAPK tumor-suppression pathways and impacting the biophysical characteristics of the structure that facilitates Gα-subunit binding. This study provides the theoretical basis for further experimental investigation required to confirm that the D125V mutation in OR2T7 is not a passenger mutation. With validation, the aforementioned mutation could represent an important prognostic marker and a potential therapeutic target for glioblastoma.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Proteína Quinase 14 Ativada por Mitógeno , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Caspase 14/genética , Caspase 14/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , PrognósticoRESUMO
OBJECTIVE: To analyze and screen the key genes of sepsis secondary to pulmonary infection by bioinformatics, and to provide theoretical basis for the basic research of the disease and find an ideal animal model program. METHODS: Experiment 1 (bioinformatics analysis): gene expression data sets of pulmonary infection secondary sepsis patients and multiple sepsis animal models were screened by Gene Expression Omnibus (GEO) Database, and gene differences were analyzed by R software. Differential genes were analyzed by gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Correlation analysis was conducted between differential genes and clinical symptoms in the data set of pulmonary infection secondary sepsis, and the correlation heat map between differential genes and clinical symptoms was drawn. Key genes were screened by weighted gene co-expression network analysis (WGCNA) and protein-protein interaction network analysis (PPIN) clustering. Experiment 2 (sepsis animal model preparation): male mice weighing 21-25 g were randomly divided into the key genes group and the control (Sham) group. And cecal ligation and puncture (CLP) was used to establish mouse sepsis model, while the mice in sham group were performed by exposure of cecum. And all the mice were scarified 24 hours after surgery to extract the total RNA from lung tissue, real time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression of key genes. RESULTS: Experiment 1 (bioinformatics analysis): 319 differential genes were showed by GSE 134364 and GSE 65682 data set analysis of pulmonary infection secondary sepsis. And there was no genetic difference between community acquired pneumonia (CAP) and hospital acquired pneumonia (HAP) in patients with pulmonary infection secondary to sepsis. Obvious differences existed between differential genes in animal models, and there was no common differential gene. Differential genes in patients and animal models were similarly enriched in GO function, mainly in cell differentiation, regulation of cell process, and regulation of cellular response to stimuli, there were significant differences in pathway enrichment, among which, CLP animal models showed higher consistency with patients. The key genes obtained by WGCNA and PPIN analysis were MAPK14, NLRC4 and LCN2. Experiment 2 (sepsis animal model preparation): animal experiment results showed that the mRNA expressions of MAPK14, NLRC4 and LCN2 in lung tissue of CLP model mice were significantly up-regulated compared with the sham group. CONCLUSIONS: MAPK14, NLRC4 and LCN2 are key genes involved in the regulation of biological processes of pulmonary sepsis secondary to infection, and are potential research directions of this disease. What's more, CLP animal model can better reflect the biological characteristics of patients with pulmonary infection secondary sepsis, and is one of the ideal animal model schemes for pulmonary infection secondary sepsis.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno , Pneumonia , Sepse , Animais , Humanos , Masculino , Camundongos , Biologia Computacional , Modelos Animais de Doenças , Expressão Gênica , Proteína Quinase 14 Ativada por Mitógeno/genética , Pneumonia/genética , RNA Mensageiro/metabolismo , Sepse/genética , Sepse/metabolismoRESUMO
Sepsis is a life-threatening medical condition caused by a dysregulated host response to infection. Recent studies have found that the expression of miRNAs is associated with the pathogenesis of sepsis and septic shock. Our study aimed to reveal which miRNAs may be involved in the dysregulated immune response in sepsis and how these miRNAs interact with transcription factors (TFs) using a computational approach with in vitro validation studies. To determine the network of TFs, miRNAs, and target genes involved in sepsis, GEO datasets GSE94717 and GSE131761 were used to identify differentially expressed miRNAs and DEGs. TargetScan and miRWalk databases were used to predict biological targets that overlap with the identified DEGs of differentially expressed miRNAs. The TransmiR database was used to predict the differential miRNA TFs that overlap with the identified DEGs. The TF-miRNA-mRNA network was constructed and visualized. Finally, qRT-PCR was used to verify the expression of TFs and miRNA in HUVECs. Between the healthy and sepsis groups, there were 146 upregulated and 98 downregulated DEGs in the GSE131761 dataset, and there were 1 upregulated and 183 downregulated DEMs in the GSE94717 dataset. A regulatory network of the TF-miRna target genes was established. According to the experimental results, RUNX3 was found to be downregulated while MAPK14 was upregulated, which corroborates the result of the computational expression analysis. In a HUVECs model, miR-19b-1-5p and miR-5009-5p were found to be significantly downregulated. Other TFs and miRNAs did not correlate with our bioinformatics expression analysis. We constructed a TF-miRNA-target gene regulatory network and identified potential treatment targets RUNX3, MAPK14, miR-19b-1-5p, and miR-5009-5p. This information provides an initial basis for understanding the complex sepsis regulatory mechanisms.
Assuntos
MicroRNAs , Proteína Quinase 14 Ativada por Mitógeno , Sepse , Fatores de Transcrição , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , MicroRNAs/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , RNA Mensageiro/genética , Sepse/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Among the body systems, the immune system plays a fundamental role in the pathophysiology of sepsis. The effects of immunogenomic and immune cell infiltration in sepsis were still not been systematically understood. Based on modified Lasso penalized regression and RF, 8 DEIRGs (ADM, CX3CR1, DEFA4, HLA-DPA1, MAPK14, ORM1, RETN, and SLPI) were combined to construct an IRG classifier. In the discovery cohort, IRG classifier exhibited superior diagnostic efficacy and performed better in predicting mortality than clinical characteristics or MARS/SRS endotypes. Encouragingly, similar results were observed in the ArrayExpress databases. The use of hydrocortisone in IRG high-risk subgroup was associated with increased risk of mortality. In IRG low-risk phenotypes, NK cells, T helper cells, and infiltrating lymphocyte (IL) are significantly richer, while T cells regulatory (Tregs) and myeloid-derived suppressor cells (MDSC) are more abundant in IRG high-risk phenotypes. IRG score were significantly negatively correlated with Cytokine cytokine receptor interaction (CCR) and human leukocyte antigen (HLA). Between the IRG subgroups, the expression levels of several cytokines (IL-10, IFNG, TNF) were significantly different, and IRG score was significantly positively correlated with ratio of IL-10/TNF. Results of qRT-PCR validated that higher expression level of ADM, DEFA4, MAPK14, ORM1, RETN, and SLPI as well as lower expression level of CX3CR1 and HLA-DPA1 in sepsis samples compared to control sample. A diagnostic and prognostic model, namely IRG classifier, was established based on 8 IRGs that is closely correlated with responses to hydrocortisone and immunosuppression status and might facilitate personalized counseling for specific therapy.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno , Sepse , Biomarcadores Tumorais/genética , Diagnóstico Precoce , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hidrocortisona , Terapia de Imunossupressão , Interleucina-10/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Prognóstico , Sepse/diagnóstico , Sepse/genética , Microambiente TumoralRESUMO
Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19.
Assuntos
Biomarcadores/sangue , COVID-19/patologia , Proteoma/análise , Adulto , Proteínas Sanguíneas/metabolismo , COVID-19/sangue , COVID-19/virologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Humanos , Influenza Humana/sangue , Influenza Humana/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Análise de Componente Principal , SARS-CoV-2/isolamento & purificação , Sepse/sangue , Sepse/patologia , Índice de Gravidade de Doença , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
BACKGROUND Heat-clearing and detoxifying herbs (HDHs) play an important role in the prevention and treatment of coronavirus infection. However, their mechanism of action needs further study. This study aimed to explore the anti-coronavirus basis and mechanism of HDHs. MATERIAL AND METHODS Database mining was performed on 7 HDHs. Core ingredients and targets were screened according to ADME rules combined with Neighborhood, Co-occurrence, Co-expression, and other algorithms. GO enrichment and KEGG pathway analyses were performed using the R language. Finally, high-throughput molecular docking was used for verification. RESULTS HDHs mainly acts on NOS3, EGFR, IL-6, MAPK8, PTGS2, MAPK14, NFKB1, and CASP3 through quercetin, luteolin, wogonin, indirubin alkaloids, ß-sitosterol, and isolariciresinol. These targets are mainly involved in the regulation of biological processes such as inflammation, activation of MAPK activity, and positive regulation of NF-kappaB transcription factor activity. Pathway analysis further revealed that the pathways regulated by these targets mainly include: signaling pathways related to viral and bacterial infections such as tuberculosis, influenza A, Ras signaling pathways; inflammation-related pathways such as the TLR, TNF, MAPK, and HIF-1 signaling pathways; and immune-related pathways such as NOD receptor signaling pathways. These pathways play a synergistic role in inhibiting lung inflammation and regulating immunity and antiviral activity. CONCLUSIONS HDHs play a role in the treatment of coronavirus infection by regulating the body's immunity, fighting inflammation, and antiviral activities, suggesting a molecular basis and new strategies for the treatment of COVID-19 and a foundation for the screening of new antiviral drugs.
Assuntos
Tratamento Farmacológico da COVID-19 , Coronavirus/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Alcaloides/química , Alcaloides/farmacologia , Caspase 3/efeitos dos fármacos , Caspase 3/genética , Coronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Bases de Dados de Produtos Farmacêuticos , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/uso terapêutico , Flavanonas/química , Flavanonas/farmacologia , Humanos , Indóis/química , Indóis/farmacologia , Interleucina-6/genética , Lignina/química , Lignina/farmacologia , Luteolina/química , Luteolina/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 8 Ativada por Mitógeno/genética , Simulação de Acoplamento Molecular , Subunidade p50 de NF-kappa B/efeitos dos fármacos , Subunidade p50 de NF-kappa B/genética , Naftóis/química , Naftóis/farmacologia , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/genética , Mapas de Interação de Proteínas , Quercetina/química , Quercetina/farmacologia , SARS-CoV-2/metabolismo , Transdução de Sinais , Sitosteroides/química , Sitosteroides/farmacologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
BACKGROUND: Prolactinoma is a functional pituitary adenoma that secretes excessive prolactin. Dopamine agonists (DAs) such as bromocriptine (BRC) are the first-line treatment for prolactinomas, but the resistance rate is increasing year by year, creating a clinical challenge. Therefore, it is urgent to explore the molecular mechanism of bromocriptine resistance in prolactinomas. Activation of the P38 MAPK pathway affects multidrug resistance in tumours. Our previous studies have demonstrated that inhibiting MAPK14 can suppress the occurrence of prolactinoma, but the role of MAPK11/12/13/14 (p38 MAPK) signalling in dopamine agonist-resistant prolactinomas is still unclear. METHODS: A prolactinoma rat model was established to determine the effect of bromocriptine on MAPK11/12/13/14 signalling. DA-resistant GH3 cells and DA-sensitive MMQ cells were used, and the role of MAPK11/12/13/14 in bromocriptine-resistant prolactinomas was preliminarily verified by western blot, RT-qPCR, ELISA, flow cytometry and CCK-8 experiments. The effects of MAPK11 or MAPK14 on bromocriptine-resistant prolactinomas were further verified by siRNA transfection experiments. RESULTS: Bromocriptine was used to treat rat prolactinoma by upregulating DRD2 expression and downregulating the expression level of MAPK11/12/13/14 in vivo experiments. The in vitro experiments showed that GH3 cells are resistant to bromocriptine and that MMQ cells are sensitive to bromocriptine. Bromocriptine could significantly reduce the expression of MAPK12 and MAPK13 in GH3 cells and MMQ cells. Bromocriptine could significantly reduce the expression of MAPK11, MAPK14, NF-κB p65 and Bcl2 in MMQ but had no effect on MAPK11, MAPK14, NF-κB p65 and Bcl2 in GH3 cells. In addition, knockdown of MAPK11 and MAPK14 in GH3 cells by siRNA transfection reversed the resistance of GH3 cells to bromocriptine, and haloperidol (HAL) blocked the inhibitory effect of bromocriptine on MAPK14, MAPK11, and PRL in MMQ cells. Our findings show that MAPK11 and MAPK14 proteins are involved in bromocriptine resistance in prolactinomas. CONCLUSION: Bromocriptine reduces the expression of MAPK11/12/13/14 in prolactinomas, and MAPK11 and MAPK14 are involved in bromocriptine resistance in prolactinomas by regulating apoptosis. Reducing the expression of MAPK11 or MAPK14 can reverse bromocriptine resistance in prolactinomas.
Assuntos
Agonistas de Dopamina/uso terapêutico , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/enzimologia , Prolactinoma/tratamento farmacológico , Prolactinoma/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Apoptose , Bromocriptina/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistência a Medicamentos , Estradiol/administração & dosagem , Estradiol/análogos & derivados , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Quinase 11 Ativada por Mitógeno/genética , Proteína Quinase 12 Ativada por Mitógeno/genética , Proteína Quinase 13 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Prolactina/genética , Prolactinoma/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/genética , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the presence of the BCR-ABL oncogene. Therapeutic regimens with tyrosine kinase inhibitors (TKIs) specifically targeting BCR-ABL have greatly improved overall survival of CML. However, drug intolerance and related toxicity remain. Combined therapy is effective in reducing drug magnitude while increasing therapeutic efficacy and, thus, lowers undesired adverse side effects. The p38 MAPK activity is critically linked to the pathogenesis of a number of diseases including hematopoietic diseases; however, the role of each isozyme in CML and TKI-mediated effects is still elusive. In this study, we used specific gene knockdown to clearly demonstrate that the deficiency of p38α greatly enhanced the therapeutic efficacy in growth suppression and cytotoxicity of TKIs, first-generation imatinib, and second generation dasatinib by approximately 2.5-3.0-fold in BCR-ABL-positive CML-derived leukemia K562 and KMB5 cells. Knockdown of p38ß, which displays the most sequence similarity to p38α, exerted distinct and opposite effects on the TKI-mediated therapeutic efficacy. These results show the importance of isotype-specific intervention in enhancing the therapeutic efficacy of TKI. A highly specific p38α inhibitor, TAK715, also significantly enhanced the imatinib- and dasatinib-mediated therapeutic efficacy, supporting the feasibility of p38α deficiency in future clinic application. Taken together, our results demonstrated that p38α is a promising target for combined therapy with BCR-ABL-targeting tyrosine kinase inhibitors for future application to increase therapeutic efficacy.
Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteína Quinase 14 Ativada por Mitógeno/genética , Terapia Combinada , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Terapia Genética , Humanos , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/deficiência , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Orchids are basically ornamental, and biological functions are seldom evaluated. This research investigated the effects of Acampe ochracea methanol extract (AOME) in ameliorating the paracetamol (PCM) induced liver injury in Wistar albino rats, evaluating its phytochemical status through UPLC-qTOF-MS analysis. With molecular docking and network pharmacology, virtual screening verified the inevitable interactions between the UPLC-qTOF-MS-characterized compounds and hepatoprotective drug receptors. The AOME has explicit a dose-dependent decrease of liver enzymes acid phosphatase (ACP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), total bilirubin, as well as an increase of serum total protein and antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSH) with a virtual normalization (p < 0.05-p < 0.001) and the values were almost equivalent to the reference drug silymarin. After pretreatment with AOME, PCM-induced malondialdehyde (MDA) levels were considerably decreased (p < 0.001). Histopathological examinations corroborated the functional and biochemical findings. The AOME upregulated the genes involved in antioxidative (CAT, SOD, ß-actin, PON1, and PFK1) and hepatoprotective mechanisms in PCM intoxicated rats. An array of 103 compounds has been identified from AOME through UPLC-qTOF-MS analysis. The detected compounds were substantially related to the targets of several liver proteins and antioxidative enzymes, according to an in silico study. Virtual prediction by SwissADME and admetSAR showed that AOME has drug-like, non-toxic, and potential pharmacological activities in hepatic damage. Furthermore, VEGFA, CYP19A1, MAPK14, ESR1, and PPARG genes interact with target compounds impacting the significant biological actions to recover PCM-induced liver damage.