RESUMEN
Disruption of fetal growth results in severe consequences to human health, including increased fetal and neonatal morbidity and mortality, as well as potential lifelong health problems. Molecular mechanisms promoting fetal growth represent potential therapeutic strategies to treat and/or prevent fetal growth restriction (FGR). Here, we identify a previously unknown role for the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) in promoting fetal and placental growth. We demonstrate that inactivation of MAP3K4 kinase activity causes FGR due in part to placental insufficiency. Significantly, MAP3K4 kinase-inactive mice display highly penetrant lethality prior to weaning and persistent growth reduction of surviving adults. Additionally, we elucidate molecular mechanisms by which MAP3K4 promotes growth through control of the insulin-like growth factor 1 receptor (IGF1R), insulin receptor (IR), and Akt signaling pathway. Specifically, MAP3K4 kinase inactivation in trophoblast stem (TS) cells results in reduced IGF1R and IR expression and decreased Akt activation. We observe these changes in TS cells also occur in differentiated trophoblasts created through in vitro differentiation of cultured TS cells and in vivo in placental tissues formed by TS cells. Furthermore, we show that MAP3K4 controls this pathway by promoting Igf1r transcript expression in TS cells through activation of CREB-binding protein (CBP). In the MAP3K4 kinase-inactive TS cells, Igf1r transcripts are repressed because of reduced CBP activity and increased histone deacetylase 6 expression and activity. Together, these data demonstrate a critical role for MAP3K4 in promoting fetal and placental growth by controlling the activity of the IGF1R/IR and Akt signaling pathway.
Asunto(s)
Desarrollo Fetal , MAP Quinasa Quinasa Quinasa 4 , Placenta , Placentación , Receptor IGF Tipo 1 , Receptor de Insulina , Adulto , Animales , Proteína de Unión a CREB/metabolismo , Femenino , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/metabolismo , Histona Desacetilasa 6/metabolismo , Humanos , MAP Quinasa Quinasa Quinasa 4/genética , MAP Quinasa Quinasa Quinasa 4/metabolismo , Ratones , Placenta/enzimología , Embarazo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transducción de SeñalRESUMEN
Schizophrenia stands out as one of the most devastating psychiatric disorders. Previous findings have shown that schizophrenia is a polygenic genetic disorder. Thus, abnormal neurodevelopment and neurogenesis may be associated with the etiology of schizophrenia, so genes which affect these processes may be potential candidate genes of schizophrenia. Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) gene is a member of the mitogen-activated protein kinase family. Taking into account previous findings, MAP3K4 plays a crucial role in the fundamental pathology of various nervous system diseases. In the present study, we aim to explore the association of MAP3K4 and schizophrenia in an independent case-control sample including 627 schizophrenic patients and 1175 healthy controls from a Northeast Chinese Han population. Both the allelic and genotypic association analyses showed that 6 SNPs in MAP3K4 were significantly associated with schizophrenia (rs590988, rs625977, rs9295134, rs12110787, rs1001808 and rs9355870). After rigorous Bonferroni correction, 4 SNPs (rs9295134, rs12110787, rs1001808 and rs9355870) were still significantly associated with the disease. The haplotype composed of these four SNPs also showed significantly global and individual association with schizophrenia. These results suggest that MAP3K4 is a susceptibility gene for schizophrenia in the Northeast Chinese Han population.
Asunto(s)
MAP Quinasa Quinasa Quinasa 4/genética , Esquizofrenia , Estudios de Casos y Controles , China/epidemiología , Predisposición Genética a la Enfermedad/genética , Genotipo , Haplotipos , Humanos , Polimorfismo de Nucleótido Simple/genética , Esquizofrenia/genéticaRESUMEN
Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) is a multifunctional mediator of the conserved MAPK signaling pathway that plays essential roles in the regulation of immune responses in mammals. However, the function of teleost MAP3K4s in innate immunity, especially in the intestinal immune system, is still poorly understood. In the current study, we identified a fish MAP3K4 homolog (CiMAP3K4) in Ctenopharyngodon idella as well as its immune function in intestine following bacterial infection in vivo and in vitro. The open reading frame (ORF) of CiMAP3K4 encodes putative peptide of 1544 amino acids containing a predicted serine/threonine protein kinase (S_TKc) domain with high identity with other fish MAP3K4s. Phylogenetic analysis revealed the CiMAP3K4 belonged to the fish cluster and showed the closest relationship to Pimephales promelas. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMAP3K4 transcripts were widely distributed in all tested tissues, especially with high expression in the muscle and intestine of healthy grass carp. In vitro, CiMAP3K4 gene expression was upregulated by bacterial PAMPs (lipolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) and muramyl dipeptide (MDP)) and pathogens (Aeromonas hydrophila and Aeromonas veronii) in primary intestinal cells. In vivo, the mRNA expression levels of CiMAP3K4 in the intestine were significantly induced by bacterial MDP challenge in a time-dependent manner; however, this effect could be inhibited by the bioactive dipeptides ß-alanyl-l-histidine (carnosine) and alanyl-glutamine (Ala-Gln). Moreover, CiMAP3K4 was located primarily in the cytoplasm, and its overexpression increased the transcriptional activity of AP-1 in HEK293T cells. Collectively, these results suggested that CiMAP3K4 might play an important role in the intestinal immune response to bacterial infections, which paves the way for a better understanding of the intestinal immune system of grass carp.
Asunto(s)
Carpas , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Bacterias Gramnegativas , MAP Quinasa Quinasa Quinasa 4 , Aeromonas hydrophila , Animales , Carpas/genética , Carpas/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Proteínas de Peces/genética , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Células HEK293 , Humanos , Inmunidad Innata/genética , Intestinos/inmunología , Intestinos/microbiología , MAP Quinasa Quinasa Quinasa 4/genética , FilogeniaRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are house-keeping enzymes that are essential for protein synthesis. However, it has become increasingly evident that some aaRSs also have non-translational functions. Here we report the identification of a non-translational function of threonyl-tRNA synthetase (ThrRS) in myogenic differentiation. We find that ThrRS negatively regulates myoblast differentiation in vitro and injury-induced skeletal muscle regeneration in vivo. This function is independent of amino acid binding or aminoacylation activity of ThrRS, and knockdown of ThrRS leads to enhanced differentiation without affecting the global protein synthesis rate. Furthermore, we show that the non-catalytic new domains (UNE-T and TGS) of ThrRS are both necessary and sufficient for the myogenic function. In searching for a molecular mechanism of this new function, we find the kinase JNK to be a downstream target of ThrRS. Our data further reveal MEKK4 and MKK4 as upstream regulators of JNK in myogenesis and the MEKK4-MKK4-JNK pathway to be a mediator of the myogenic function of ThrRS. Finally, we show that ThrRS physically interacts with Axin1, disrupts Axin1-MEKK4 interaction and consequently inhibits JNK signaling. In conclusion, we uncover a non-translational function for ThrRS in the maintenance of homeostasis of skeletal myogenesis and identify the Axin1-MEKK4-MKK4-JNK signaling axis to be an immediate target of ThrRS action.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Desarrollo de Músculos , Treonina-ARNt Ligasa/metabolismo , Animales , Proteína Axina/metabolismo , Femenino , MAP Quinasa Quinasa 4/metabolismo , MAP Quinasa Quinasa Quinasa 4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Biosíntesis de Proteínas , Dominios Proteicos , Treonina-ARNt Ligasa/químicaRESUMEN
Sex determination requires the commitment of bipotential gonads to either a testis or an ovarian fate. Gene deletion of the kinase Map3k4 results in gonadal sex reversal in XY mice, and transgenic re-expression of Map3k4 rescues the sex reversal phenotype. Map3k4 encodes a large, multi-functional protein possessing a kinase domain and several, additional protein-protein interaction domains. Although MAP3K4 plays a critical role in male gonadal sex determination, it is unknown if the kinase activity of MAP3K4 is required. Here, we use mice expressing full-length, kinase-inactive MAP3K4 from the endogenous Map3k4 locus to examine the requirement of MAP3K4 kinase activity in sex determination. Although homozygous kinase-inactivation of MAP3K4 (Map3k4KI/KI) is lethal, a small fraction survive to adulthood. We show Map3k4KI/KI adults exhibit a 4:1 female-biased sex ratio. Many adult Map3k4KI/KI phenotypic females have a Y chromosome. XY Map3k4KI/KI adults with sex reversal display female mating behavior, but do not give rise to offspring. Reproductive organs are overtly female, but there is a broad spectrum of ovarian phenotypes, including ovarian absence, primitive ovaries, reduced ovarian size, and ovaries having follicles in all stages of development. Further, XY Map3k4KI/KI adults are smaller than either male or female Map3k4WT/WT mice. Examination of the critical stage of gonadal sex determination at E11.5 shows that loss of MAP3K4 kinase activity results in the loss of Sry expression in XY Map3k4KI/KI embryos, indicating embryonic male gonadal sex reversal. Together, these findings demonstrate the essential role for kinase activity of MAP3K4 in male gonadal sex determination.
Asunto(s)
MAP Quinasa Quinasa Quinasa 4/genética , Ratones/genética , Ovario/embriología , Procesos de Determinación del Sexo/genética , Testículo/embriología , Animales , Femenino , MAP Quinasa Quinasa Quinasa 4/metabolismo , Masculino , Ratones/embriologíaRESUMEN
Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growthfactor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAFmutant CSPG4positive or negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patientderived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4positive cells as compared to pretherapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drugresistance in melanoma cells.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Melanoma/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Línea Celular Tumoral , Proteoglicanos Tipo Condroitín Sulfato/genética , Progresión de la Enfermedad , Regulación hacia Abajo , Resistencia a Antineoplásicos , Humanos , MAP Quinasa Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa Quinasa 4/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas de la Membrana/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis. Long non-coding RNAs (lncRNAs) have emerged as essential inflammation regulators. We identify a novel lncRNA termed lncRNA-MAP3K4 that is enriched in the vessel wall and regulates vascular inflammation. In the aortic intima, lncRNA-MAP3K4 expression was reduced by 50% during the progression of atherosclerosis (chronic inflammation) and 70% during endotoxemia (acute inflammation). lncRNA-MAP3K4 knockdown reduced the expression of key inflammatory factors (eg, ICAM-1, E-selectin, MCP-1) in endothelial cells or vascular smooth muscle cells and decreased monocytes adhesion to endothelium, as well as reducing TNF-α, IL-1ß, COX2 expression in macrophages. Mechanistically, lncRNA-MAP3K4 regulates inflammation through the p38 MAPK signaling pathway. lncRNA-MAP3K4 shares a bidirectional promoter with MAP3K4, an upstream regulator of the MAPK signaling pathway, and regulates its transcription in cis. lncRNA-MAP3K4 and MAP3K4 show coordinated expression in response to inflammation in vivo and in vitro. Similar to lncRNA-MAP3K4, MAP3K4 knockdown reduced the expression of inflammatory factors in several different vascular cells. Furthermore, lncRNA-MAP3K4 and MAP3K4 knockdown showed cooperativity in reducing inflammation in endothelial cells. Collectively, these findings unveil the role of a novel lncRNA in vascular inflammation by cis-regulating MAP3K4 via a p38 MAPK pathway.
Asunto(s)
Regulación de la Expresión Génica , MAP Quinasa Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas , ARN Largo no Codificante/metabolismo , Vasculitis/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , MAP Quinasa Quinasa Quinasa 4/genética , Ratones , ARN Largo no Codificante/genética , Vasculitis/genética , Vasculitis/patología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Non-coding mutations can create splice sites, however the true extent of how such somatic non-coding mutations affect RNA splicing are largely unexplored. Here we use the MiSplice pipeline to analyze 783 cancer cases with WGS data and 9494 cases with WES data, discovering 562 non-coding mutations that lead to splicing alterations. Notably, most of these mutations create new exons. Introns associated with new exon creation are significantly larger than the genome-wide average intron size. We find that some mutation-induced splicing alterations are located in genes important in tumorigenesis (ATRX, BCOR, CDKN2B, MAP3K1, MAP3K4, MDM2, SMAD4, STK11, TP53 etc.), often leading to truncated proteins and affecting gene expression. The pattern emerging from these exon-creating mutations suggests that splice sites created by non-coding mutations interact with pre-existing potential splice sites that originally lacked a suitable splicing pair to induce new exon formation. Our study suggests the importance of investigating biological and clinical consequences of noncoding splice-inducing mutations that were previously neglected by conventional annotation pipelines. MiSplice will be useful for automatically annotating the splicing impact of coding and non-coding mutations in future large-scale analyses.
Asunto(s)
Neoplasias/genética , Precursores del ARN/genética , Sitios de Empalme de ARN , Empalme del ARN , Quinasas de la Proteína-Quinasa Activada por el AMP , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Bases de Datos Genéticas , Exones , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Intrones , Quinasa 1 de Quinasa de Quinasa MAP/genética , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , MAP Quinasa Quinasa Quinasa 4/genética , MAP Quinasa Quinasa Quinasa 4/metabolismo , Mutación , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN no Traducido , RNA-Seq , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Secuenciación del Exoma , Proteína Nuclear Ligada al Cromosoma X/genética , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
Coordinated gene expression is required for phenotypic switching between epithelial and mesenchymal phenotypes during normal development and in disease states. Trophoblast stem (TS) cells undergo epithelial-mesenchymal transition (EMT) during implantation and placentation. Mechanisms coordinating gene expression during these processes are poorly understood. We have previously demonstrated that MAP3K4-regulated chromatin modifiers CBP and HDAC6 each regulate thousands of genes during EMT in TS cells. Here we show that CBP and HDAC6 coordinate expression of only 183 genes predicted to be critical regulators of phenotypic switching. The highest-ranking co-regulated gene is the NF-κB family member Rel. Although NF-κB is primarily regulated post-transcriptionally, CBP and HDAC6 control Rel transcript levels by binding Rel regulatory regions and controlling histone acetylation. REL re-expression in mesenchymal-like TS cells induces a mesenchymal-epithelial transition. Importantly, REL forms a feedback loop, blocking HDAC6 expression and nuclear localization. Together, our work defines a developmental program coordinating phenotypic switching.
Asunto(s)
Regulación de la Expresión Génica , Histona Desacetilasa 6/metabolismo , MAP Quinasa Quinasa Quinasa 4/metabolismo , Proteínas Oncogénicas v-rel/genética , Fragmentos de Péptidos/metabolismo , Fenotipo , Sialoglicoproteínas/metabolismo , Animales , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Masculino , Ratones , Modelos Biológicos , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-met/metabolismo , Células Madre/metabolismo , Factores de TranscripciónRESUMEN
Autophagy and apoptosis, which are important processes for host immunity, are commonly exploited by viruses to facilitate their survival. However, to the best of our knowledge, very few studies have researched the mechanisms of action of the autophagic and apoptotic signaling pathways following viral infection. Thus, the present study aimed to investigate the mechanisms of action of growth arrest and DNA-damage-inducible ß (GADD45ß), an important resistance gene involved in the host resistance to ALV-J. Both ALV-J infection and the overexpression of GADD45ß inhibited autophagy during the early stages, which prevented the autophagosomes from binding to the lysosomes and resulted in an incomplete autophagic flux. Notably, GADD45ß was discovered to interact with MEKK4 in DF-1 cells. The genetic knockdown of GADD45ß and MEKK4 using small interfering RNA-affected ALV-J infection, which suggested that ALV-J may promote the binding of GADD45ß to MEKK4 to activate the p38MAPK signaling pathway, which subsequently inhibits autophagy. Furthermore, ALV-J was revealed to affect the autophagic pathway prior to affecting the apoptotic pathway. In conclusion, to the best of our knowledge, the present study was the first to investigate the combined effects of ALV-J infection on autophagy and apoptosis, and to suggest that ALV-J inhibits autophagy via the GADD45ß/MEKK4/p38MAPK signaling pathway.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Autofagia/fisiología , Virus de la Leucosis Aviar/metabolismo , Animales , Apoptosis/fisiología , Virus de la Leucosis Aviar/genética , Virus de la Leucosis Aviar/patogenicidad , Línea Celular , Embrión de Pollo , Pollos/genética , Interacciones Huésped-Patógeno/fisiología , MAP Quinasa Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Interleukin (IL)1ß is a key promotor in the pathogenesis of temporomandibular joint osteoarthritis. Differentiation of stem cells to cartilage is a crucial repair mechanism of articular cartilage damage, and IL1ß has been reported to impede the differentiation by upregulating the secretion of IL6, an important inflammatory factor. Long noncoding RNAs (lncRNAs) regulate a number of physiological and pathological processes, but whether lncRNA AK094629 contributes to the IL1ß mediated induction of inflammation remains unclear. Therefore, the aim of the present study was to investigate the effect of AK094629 on IL1ßinduced IL6 expression in synovialderived mesenchymal stem cells (SMSCs) of the temporomandibular joints. The results of the present study demonstrated that the expression of AK094629 in the synovial tissue of patients with osteoarthritis was positively correlated with IL1ß. In addition, IL1ß upregulated the expression of AK094629 in the SMSCs in vitro, and AK094629 knockdown inhibited the IL1ß mediated upregulation of IL6. The present study also demonstrated that AK094629 knockdown downregulated the expression of the mitogenactivated protein kinase kinase kinase 4 (MAP3K4), which is upregulated by IL1ß, whereas knockdown of MAP3K4 did not affect the expression of AK094629, but reversed the upregulation of IL6 in SMSCs. In conclusion, AK094629 knockdown attenuated the expression of IL1ßregulated IL6 in the SMSCs of the temporomandibular joint by inhibiting MAP3K4. Therefore, AK094629 may be a potential novel therapeutic target for the treatment of temporomandibular joint osteoarthritis.
Asunto(s)
Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Células Madre Mesenquimatosas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Membrana Sinovial/metabolismo , Articulación Temporomandibular/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-6/genética , MAP Quinasa Quinasa Quinasa 4/genética , MAP Quinasa Quinasa Quinasa 4/metabolismo , Células Madre Mesenquimatosas/patología , Osteoartritis/etiología , Osteoartritis/metabolismo , Osteoartritis/patología , Membrana Sinovial/citología , Articulación Temporomandibular/patología , Trastornos de la Articulación Temporomandibular/etiología , Trastornos de la Articulación Temporomandibular/metabolismo , Trastornos de la Articulación Temporomandibular/patología , Activación Transcripcional/genética , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Macrolide antibiotics, such as erythromycin and josamycin, are natural polyketide products harboring 14- to 16-membered macrocyclic lactone rings to which various sugars are attached. These antibiotics are used extensively in the clinic because of their ability to inhibit bacterial protein synthesis. More recently, some macrolides have been shown to also possess anti-inflammatory and other therapeutic activities in mammalian cells. To better understand the targets and effects of this drug class in mammalian cells, we used a genome-wide shRNA screen in K562 cancer cells to identify genes that modulate cellular sensitivity to josamycin. Among the most sensitizing hits were proteins involved in mitochondrial translation and the mitochondrial unfolded protein response, glycolysis, and the mitogen-activated protein kinase signaling cascade. Further analysis revealed that cells treated with josamycin or other antibacterial agents exhibited impaired oxidative phosphorylation and metabolic shifts to glycolysis. Interestingly, we observed that knockdown of the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) gene, which contributes to p38 mitogen-activated protein kinase signaling, sensitized cells only to josamycin but not to other antibacterial agents. There is a growing interest in better characterizing the therapeutic effects and toxicities of antibiotics in mammalian cells to guide new applications in both cellular and clinical studies. To our knowledge, this is the first report of an unbiased genome-wide screen to investigate the effects of a clinically used antibiotic on human cells.
Asunto(s)
Antibacterianos/farmacología , MAP Quinasa Quinasa Quinasa 4/genética , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Animales , Antibacterianos/efectos adversos , Farmacorresistencia Microbiana/efectos de los fármacos , Eritromicina/efectos adversos , Eritromicina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Josamicina/efectos adversos , Josamicina/farmacología , Células K562 , MAP Quinasa Quinasa Quinasa 4/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrólidos/efectos adversos , Macrólidos/farmacología , Proteínas Quinasas Activadas por Mitógenos/genética , Fosforilación Oxidativa/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/efectos adversos , Inhibidores de la Síntesis de la Proteína/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Many studies have verified that microRNAs contribute a lot to neuropathic pain progression. Furthermore, nerve-related inflammatory cytokines play vital roles in neuropathic pain progression. miR-183 has been identified to have a common relationship with multiple pathological diseases. However, the potential effects of miR-183 in the process of neuropathic pain remain undetermined. Therefore, we performed the current study with the purpose of finding the functions of miR-183 in neuropathic pain progression using a chronic sciatic nerve injury (CCI) rat model. We demonstrated that miR-183 expression levels were evidently reduced in CCI rats in contrast with the control group. Overexpression of miR-183 produced significant relief of mechanical hyperalgesia, as well as thermal hyperalgesia in CCI rats. Furthermore, neuropathic pain-correlated inflammatory cytokine expression levels containing interleukin-6 (IL-6) and interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2) were obviously inhibited by upregulation of miR-183. Meanwhile, dual-luciferase reporter assays showed MAP3K4 was a direct downstream gene of miR-183. The expression levels of MAP3K4 were modulated by the increased miR-183 negatively, which lead to the downregulation of IL-6, IL-1ß, and COX-2, and then reduced neuropathic pain progression, respectively. Overall, our study pointed out that miR-183 was a part of the negative regulator which could relieve neuropathic pain by targeting MAP3K4. Thus it may provide a new clinical treatment for neuropathic pain patients clinical therapy.
Asunto(s)
MAP Quinasa Quinasa Quinasa 4/genética , MicroARNs/genética , Neuralgia/genética , Neuropatía Ciática/genética , Animales , Ciclooxigenasa 2/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hiperalgesia , Interleucina-1beta/genética , Interleucina-6/genética , Neuralgia/tratamiento farmacológico , Neuralgia/patología , Ratas , Ratas Sprague-Dawley , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , Nervio Ciático/patología , Neuropatía Ciática/tratamiento farmacológico , Neuropatía Ciática/patologíaRESUMEN
BACKGROUND: Non-syndromic intellectual disability is genetically heterogeneous with dominant, recessive and complex forms of inheritance. We have performed detailed genetic studies in a large multi-generational Swedish family, including several members diagnosed with non-syndromic intellectual disability. Linkage analysis was performed on 22 family members, nine affected with mild to moderate intellectual disability and 13 unaffected family members. METHODS: Family members were analyzed with Affymetrix Genome-Wide Human SNP Array 6.0 and the genetic data was used to detect copy number variation and to perform genome wide linkage analysis with the SNP High Throughput Linkage analysis system and the Merlin software. For the exome sequencing, the samples were prepared using the Sure Select Human All Exon Kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced using the Ion Proton™ System. Validation of identified variants was performed with Sanger sequencing. RESULTS: The linkage analysis results indicate that intellectual disability in this family is genetically heterogeneous, with suggestive linkage found on chromosomes 1q31-q41, 4q32-q35, 6p25 and 14q24-q31 (LOD scores of 2.4, simulated p-value of 0.000003 and a simulated genome-wide p-value of 0.06). Exome sequencing was then performed in 14 family members and 7 unrelated individuals from the same region. The analysis of coding variation revealed a pathogenic and candidate variants in different branches of the family. In three patients we find a known homozygous pathogenic mutation in the Homo sapiens solute carrier family 17 member 5 (SLC17A5), causing Salla disease. We also identify a deletion overlapping KDM3B and a duplication overlapping MAP3K4 and AGPAT4, both overlapping variants previously reported in developmental disorders. CONCLUSIONS: DNA samples from the large family analyzed in this study were initially collected based on a hypothesis that affected members shared a major genetic risk factor. Our results show that a complex phenotype such as mild intellectual disability in large families from genetically isolated populations may show considerable genetic heterogeneity.
Asunto(s)
Exoma/genética , Ligamiento Genético , Discapacidad Intelectual/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Variaciones en el Número de Copia de ADN , Humanos , Discapacidad Intelectual/patología , Histona Demetilasas con Dominio de Jumonji/genética , Cariotipificación , MAP Quinasa Quinasa Quinasa 4/genética , Transportadores de Anión Orgánico/genética , Linaje , Polimorfismo de Nucleótido Simple , Suecia , Simportadores/genética , Secuenciación del ExomaRESUMEN
One of the major features of cancer is Otto Warburg's observation that many tumors have increased extracellular acidification compared to healthy tissues. Since Warburg's observation, the importance of extracellular acidification in cancer is now considered a hallmark of cancer. Human MAP3K4 functions upstream of the p38 and JNK mitogen activated protein kinases (MAPKs). Additionally, MAP3K4 is required for cell migration and extracellular acidification of breast cancer cells in response to HER2/HER3 signaling. Here, we demonstrate that GIT1 interacts with MAP3K4 by immunoprecipitation, while cellular lactate production and the capacity of MCF-7 cells for anchorage independent growth in soft agar were dependent on GIT1. Additionally, we show that activation of HER2/HER3 signaling leads to reduced expression of lactate receptor (GPR81) mRNA and that both, GIT1 and MAP3K4, are necessary for constitutive expression of GPR81 mRNA. Our study suggests that targeting downstream proteins in the HER2/HER3-induced extracellular lactate signaling pathway may be a way to inhibit the Warburg Effect to disrupt tumor growth.
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Ácido Láctico/metabolismo , MAP Quinasa Quinasa Quinasa 4/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transducción de Señal/fisiología , Microambiente Tumoral/fisiología , Animales , Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Ratones , Músculo Esquelético/metabolismo , Fosforilación , ARN MensajeroRESUMEN
BACKGROUND: Chronic Lymphocytic Leukemia (CLL) is the most frequent lymphoproliferative disorder in western countries and is characterized by a remarkable clinical heterogeneity. During the last decade, multiple genomic studies have identified a myriad of somatic events driving CLL proliferation and aggressivity. Nevertheless, and despite the mounting evidence of inherited risk for CLL development, the existence of germline variants associated with clinical outcomes has not been addressed in depth. METHODS: Exome sequencing data from control leukocytes of CLL patients involved in the International Cancer Genome Consortium (ICGC) was used for genotyping. Cox regression was used to detect variants associated with clinical outcomes. Gene and pathways level associations were also calculated. RESULTS: Single nucleotide polymorphisms in PPP4R2 and MAP3K4 were associated with earlier treatment need. A gene-level analysis evidenced a significant association of RIPK3 with both treatment need and survival. Furthermore, germline variability in pathways such as apoptosis, cell-cycle, pentose phosphate, GNα13 and Nitric oxide was associated with overall survival. CONCLUSION: Our results support the existence of inherited conditionants of CLL evolution and points towards genes and pathways that may results useful as biomarkers of disease outcome. More research is needed to validate these findings.
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Biomarcadores de Tumor/genética , Secuenciación del Exoma/métodos , Mutación de Línea Germinal , Leucemia Linfocítica Crónica de Células B/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Humanos , MAP Quinasa Quinasa Quinasa 4/genética , Masculino , Fosfoproteínas Fosfatasas/genética , Análisis de SupervivenciaRESUMEN
Missense mutations in the gene, MAP3K1, are a common cause of 46,XY gonadal dysgenesis, accounting for 15-20% of cases [Ostrer, 2014, Disorders of sex development (DSDs): an update. J. Clin. Endocrinol. Metab., 99, 1503-1509]. Functional studies demonstrated that all of these mutations cause a protein gain-of-function that alters co-factor binding and increases phosphorylation of the downstream MAP kinase pathway targets, MAPK11, MAP3K and MAPK1. This dysregulation of the MAP kinase pathway results in increased CTNNB1, increased expression of WNT4 and FOXL2 and decreased expression of SRY and SOX9. Unique and recurrent pathogenic mutations cluster in three semi-contiguous domains outside the kinase region of the protein, a newly identified N-terminal domain that shares homology with the Guanine Exchange Factor (residues Met164 to Glu231), a Plant HomeoDomain (residues Met442 to Trp495) and an ARMadillo repeat domain (residues Met566 to Glu862). Despite the presence of the mutation clusters and clinical data, there exists a dearth of mechanistic insights behind the development imbalance. In this paper, we use structural modeling and functional data of these mutations to understand alterations of the MAP3K1 protein and the effects on protein folding, binding and downstream target phosphorylation. We show that these mutations have differential effects on protein binding depending on the domains in which they occur. These mutations increase the binding of the RHOA, MAP3K4 and FRAT1 proteins and generally decrease the binding of RAC1. Thus, pathologies in MAP3K1 disrupt the balance between the pro-kinase activities of the RHOA and MAP3K4 binding partners and the inhibitory activity of RAC1.
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Trastornos del Desarrollo Sexual/genética , Quinasa 1 de Quinasa de Quinasa MAP/genética , MAP Quinasa Quinasa Quinasa 4/genética , Proteína de Unión al GTP rac1/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Dominio Armadillo/genética , Trastorno del Desarrollo Sexual 46,XY , Trastornos del Desarrollo Sexual/patología , Femenino , Proteína Forkhead Box L2/genética , Regulación de la Expresión Génica/genética , Disgenesia Gonadal 46 XY/genética , Disgenesia Gonadal 46 XY/patología , Humanos , Quinasa 1 de Quinasa de Quinasa MAP/química , MAP Quinasa Quinasa Quinasa 4/química , Sistema de Señalización de MAP Quinasas/genética , Masculino , Mutación Missense/genética , Unión Proteica/genética , Proteínas Proto-Oncogénicas/genética , Proteína de la Región Y Determinante del Sexo/genética , Proteína de Unión al GTP rac1/química , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/genéticaRESUMEN
MAPKs affect gonadal differentiation in mice and humans, but whether this applies to all mammals is as yet unknown. Thus, we investigated MAPK expression during gonadal differentiation and after treatment with oestrogen in a distantly related mammal, the marsupial tammar wallaby, using our model of oestrogen-induced gonadal sex reversal. High-throughput RNA-sequencing was carried out on gonads collected from developing tammar 2 days before birth to 8 days after birth to characterise MAPK and key sexual differentiation markers. Day 25 foetal testes were cultured for 120 h in control medium or medium supplemented with exogenous oestrogen and processed for RNA-seq to identify changes in gene expression in response to oestrogen. MAPK pathway genes in the tammar were highly conserved at the sequence and amino acid level with those of mice and humans. Marsupial MAP3K1 and MAP3K4 clustered together in a separate branch from eutherian mammals. There was a marked decrease in the expression of male-determining genes SOX9 and AMH and increase in the female marker FOXL2 in oestrogen-treated male gonads. Only MAP3K1 expression increased in male gonads in response to oestrogen while other MAPK genes remained unaffected. This study suggests that MAP3K1 can be influenced by exogenous oestrogens during gonadal differentiation in this marsupial.
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Perfilación de la Expresión Génica , Gónadas/embriología , Gónadas/enzimología , Quinasa 1 de Quinasa de Quinasa MAP/genética , MAP Quinasa Quinasa Quinasa 4/genética , Macropodidae/embriología , Macropodidae/genética , Animales , Estrógenos/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Gónadas/efectos de los fármacos , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , MAP Quinasa Quinasa Quinasa 4/metabolismo , Masculino , Filogenia , Diferenciación Sexual/efectos de los fármacos , Diferenciación Sexual/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
The age-related reduction in the function of osteoblasts plays a central role in the pathogenesis of bone loss and osteoporosis. Collagen synthesis is a primary function of differentiated osteoblasts, however, the mechanisms for age-related changes in collagen synthesis in human osteoblasts remain elusive. We use Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) analysis to exploit the transcriptional profiles of osteoblasts from young and old donors. A panel of collagen members was downregulated in aged osteoblasts, including COL12A1, COL5A1, COL5A3, COL8A1 and COL8A2. Co-expression analysis followed by GO analysis revealed that oxidoreductase activity and kinase activity were inversely correlated with collagen synthesis in osteoblasts. GESA analysis further showed that JNK signaling was upregulated in aged osteoblasts. Consistently, MAP3K4 and MAP4K2, upstream of JNK, were also increased in aged osteoblasts. Moreover, expression levels of MAP3K4 were significantly inversely correlated with levels of the collagen genes. Those transcriptomic results were further verified by examining clinical specimens of osteoporosis by immunohistochemistry. These results provide transcriptomic evidence that deregulated JNK signaling may impair collagen synthesis in osteoblasts and imply a therapeutic value of JNK inhibitors for treating osteoporosis and preventing skeletal aging by counteracting the age-related reduction in the function of osteoblasts.
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Colágeno/biosíntesis , Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas/fisiología , Osteoblastos/metabolismo , Osteoporosis/metabolismo , Adulto , Factores de Edad , Anciano , Colágeno/genética , Colágeno Tipo VIII/genética , Colágeno Tipo VIII/metabolismo , Colágeno Tipo XII/genética , Colágeno Tipo XII/metabolismo , Quinasas del Centro Germinal , Humanos , MAP Quinasa Quinasa Quinasa 4/genética , MAP Quinasa Quinasa Quinasa 4/metabolismo , Persona de Mediana Edad , Osteoblastos/fisiología , Osteoporosis/patología , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Análisis de Secuencia de ARNRESUMEN
We have recently reported that male rats given liquid fructose ingestion exhibit features of cardiometabolic abnormalities including non-obese insulin resistance with impaired insulin signaling transduction in skeletal muscle (Rattanavichit Y et al. Am J Physiol Regul Integr Comp Physiol 311: R1200-R1212, 2016). While exercise can attenuate obesity-related risks of cardiometabolic syndrome, the effectiveness and potential mechanism by which exercise modulates non-obese insulin resistance have not been fully studied. The present investigation evaluated whether regular exercise by voluntary wheel running (VWR) can reduce cardiometabolic risks induced by fructose ingestion. Moreover, the potential cellular adaptations following VWR on key signaling proteins known to influence insulin-induced glucose transport in skeletal muscle of fructose-ingested rats were investigated. Male Sprague-Dawley rats were given either water or liquid fructose (10% wt/vol) without or with access to running wheel for 6 weeks. We demonstrated that VWR restored insulin-stimulated glucose transport in the soleus muscle by improving the functionality of several signaling proteins, including insulin-stimulated IRBetaTyr1158/Tyr1162/Tyr1163 (82%), IRS-1 Tyr989 (112%), Akt Ser473 (56%), AS160 Thr642 (76%), and AS160 Ser588 (82%). These effects were accompanied by lower insulin-stimulated phosphorylation of IRS-1 Ser307 (37%) and JNK Thr183/Tyr185 (49%), without significant changes in expression of proteins in the renin-angiotensin system. Intriguingly, multiple cardiometabolic abnormalities were not observed in fructose-ingested rats with access to VWR. Collectively, this study demonstrates that the development of cardiometabolic abnormalities as well as insulin resistance of skeletal muscle and defective signaling molecules in rats induced by fructose ingestion could be opposed by VWR