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1.
Cells ; 8(8)2019 08 01.
Article in English | MEDLINE | ID: mdl-31374860

ABSTRACT

Dysregulation of receptor tyrosine kinase-induced pathways is a critical step driving the oncogenic potential of brain cancer. In this study, we investigated the role of two members of the Sprouty (Spry) family in brain cancer-derived cell lines. Using immunoblot analyses we found essential differences in the pattern of endogenous Spry3 and Spry4 expression. While Spry4 expression was mitogen-dependent and repressed in a number of cells from higher malignant brain cancers, Spry3 levels neither fluctuated in response to serum withdrawal nor were repressed in glioblastoma (GBM)-derived cell lines. In accordance to the well-known inhibitory role of Spry proteins in fibroblast growth factor (FGF)-mediated signaling, both Spry proteins were able to interfere with FGF-induced activation of the MAPK pathway although to a different extent. In response to serum solely, Spry4 exerts its role as a negative regulator of MAPK activation. Ectopic expression of Spry4 inhibited proliferation and migration of GBM-originated cells, positioning it as a tumor suppressor in brain cancer. In contrast, elevated Spry3 levels accelerated both proliferation and migration of these cell lines, while repression of Spry3 levels using shRNA caused a significant diminished growth and migration velocity rate of a GBM-derived cell line. This argues for a tumor-promoting function of Spry3 in GBMs. Based on these data we conclude that Spry3 and Spry4 fulfill different if not opposing roles within the cancerogenesis of brain malignancies.


Subject(s)
Brain Neoplasms/metabolism , Carcinogenesis/metabolism , Glioblastoma/metabolism , Intracellular Signaling Peptides and Proteins/physiology , Nerve Tissue Proteins/physiology , Cell Line, Tumor , Cell Proliferation , Humans , RNA, Small Interfering/metabolism , Signal Transduction
2.
J Chromatogr A ; 1423: 183-9, 2015 Dec 04.
Article in English | MEDLINE | ID: mdl-26554298

ABSTRACT

Nucleotide sugars, the activated forms of monosaccharides, are important metabolites involved in a multitude of cellular processes including glycosylation of xenobiotics. Especially in plants, UDP-glucose is one of the most prominent members among these nucleotide-sugars, as it is involved in the formation of glucose conjugates of xenobiotics, including mycotoxins, but also holds a central role in the interconversion of energized sugars such as the formation of UDP-glucuronic acid required for cell wall biosynthesis. Here, we present the first HILIC-LC-ESI-TQ-MS/MS method for the quantification of UDP-glucose and UDP-glucuronic acid together with the Fusarium toxin deoxynivalenol (DON) and its major plant detoxification product DON-3-O-glucoside (DON-3-Glc) utilizing a polymer-based column. For sample preparation a time-effective and straightforward 'dilute and shoot' protocol was applied. The chromatographic run time was minimized to 9min including proper column re-equilibration. In-house validation of the method verified its linear range, intra- (1-7%) and interday (8-20%) precision, instrumental LODs between 0.6 and 10ngmL(-1), selectivity and moderate matrix effects with mean recoveries of 85-103%. To prove the methods applicability, we analyzed two sets of wheat extracts obtained from different cultivars grown under standardized greenhouse conditions. The results clearly demonstrated the suitability of the developed method to quantify UDP-glucose, DON and its masked form D3G in diluted wheat extracts. We observed differing concentration levels of UDP-glucose in the two wheat cultivars showing different resistance to the severe plant disease Fusarium head blight. We propose that the higher ability to detoxify DON into DON-3-Glc might be a consequence of the higher cellular UDP-glucose pool in the resistant cultivar.


Subject(s)
Chromatography, Liquid , Glycosides/analysis , Hazard Analysis and Critical Control Points/methods , Tandem Mass Spectrometry , Triticum/chemistry , Food Contamination/analysis , Fusarium/chemistry , Glucose/analysis , Glucose/metabolism , Glucosides/analysis , Glucuronates/analysis , Hydrophobic and Hydrophilic Interactions , Mycotoxins/analysis , Trichothecenes/analysis , Triticum/microbiology , Uridine Diphosphate Glucose/analysis , Uridine Diphosphate Glucuronic Acid/analysis
3.
G3 (Bethesda) ; 5(12): 2579-92, 2015 Oct 04.
Article in English | MEDLINE | ID: mdl-26438291

ABSTRACT

Fusarium head blight is a prevalent disease of bread wheat (Triticum aestivum L.), which leads to considerable losses in yield and quality. Quantitative resistance to the causative fungus Fusarium graminearum is poorly understood. We integrated transcriptomics and metabolomics data to dissect the molecular response to the fungus and its main virulence factor, the toxin deoxynivalenol in near-isogenic lines segregating for two resistance quantitative trait loci, Fhb1 and Qfhs.ifa-5A. The data sets portrait rearrangements in the primary metabolism and the translational machinery to counter the fungus and the effects of the toxin and highlight distinct changes in the metabolism of glutamate in lines carrying Qfhs.ifa-5A. These observations are possibly due to the activity of two amino acid permeases located in the quantitative trait locus confidence interval, which may contribute to increased pathogen endurance. Mapping to the highly resolved region of Fhb1 reduced the list of candidates to few genes that are specifically expressed in presence of the quantitative trait loci and in response to the pathogen, which include a receptor-like protein kinase, a protein kinase, and an E3 ubiquitin-protein ligase. On a genome-scale level, the individual subgenomes of hexaploid wheat contribute differentially to defense. In particular, the D subgenome exhibited a pronounced response to the pathogen and contributed significantly to the overall defense response.


Subject(s)
Basal Metabolism , Genomics , Metabolome , Plant Diseases/genetics , Transcriptome , Triticum/genetics , Triticum/metabolism , Computational Biology/methods , Disease Resistance/genetics , Fusarium/physiology , Gene Expression Profiling , Gene Expression Regulation, Plant , Genomics/methods , Glutamic Acid , Host-Pathogen Interactions/genetics , Metabolic Networks and Pathways , Metabolomics , Plant Diseases/microbiology , Quantitative Trait Loci , RNA Ligase (ATP)/metabolism , Trichothecenes/toxicity , Triticum/drug effects , Triticum/microbiology , Ubiquitination
4.
BMC Genomics ; 14: 728, 2013 Oct 24.
Article in English | MEDLINE | ID: mdl-24152241

ABSTRACT

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium graminearum Schwabe is one of the most prevalent diseases of wheat (Triticum aestivum L.) and other small grain cereals. Resistance against the fungus is quantitative and more than 100 quantitative trait loci (QTL) have been described. Two well-validated and highly reproducible QTL, Fhb1 and Qfhs.ifa-5A have been widely investigated, but to date the underlying genes have not been identified. RESULTS: We have investigated a gene co-expression network activated in response to F. graminearum using RNA-seq data from near-isogenic lines, harboring either the resistant or the susceptible allele for Fhb1 and Qfhs.ifa-5A. The network identified pathogen-responsive modules, which were enriched for differentially expressed genes between genotypes or different time points after inoculation with the pathogen. Central gene analysis identified transcripts associated with either QTL within the network. Moreover, we present a detailed gene expression analysis of four gene families (glucanases, NBS-LRR, WRKY transcription factors and UDP-glycosyltransferases), which take prominent roles in the pathogen response. CONCLUSIONS: A combination of a network-driven approach and differential gene expression analysis identified genes and pathways associated with Fhb1 and Qfhs.ifa-5A. We find G-protein coupled receptor kinases and biosynthesis genes for jasmonate and ethylene earlier induced for Fhb1. Similarly, we find genes involved in the biosynthesis and metabolism of riboflavin more abundant for Qfhs.ifa-5A.


Subject(s)
Fusarium/pathogenicity , Plant Proteins/genetics , Triticum/genetics , Calcium Signaling/genetics , Diacylglycerol Kinase/genetics , Diacylglycerol Kinase/metabolism , Disease Resistance/genetics , Fusarium/genetics , Fusarium/metabolism , Genotype , Plant Proteins/metabolism , Quantitative Trait Loci , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Sequence Analysis, RNA , Transcriptome , Triticum/metabolism , Triticum/microbiology
5.
Mol Plant Pathol ; 14(8): 772-85, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23738863

ABSTRACT

Fusarium head blight, caused by Fusarium graminearum, is a devastating disease of wheat. We developed near-isogenic lines (NILs) differing in the two strongest known F. graminearum resistance quantitative trait loci (QTLs), Qfhs.ndsu-3BS (also known as resistance gene Fhb1) and Qfhs.ifa-5A, which are located on the short arm of chromosome 3B and on chromosome 5A, respectively. These NILs showing different levels of resistance were used to identify transcripts that are changed significantly in a QTL-specific manner in response to the pathogen and between mock-inoculated samples. After inoculation with F. graminearum spores, 16 transcripts showed a significantly different response for Fhb1 and 352 for Qfhs.ifa-5A. Notably, we identified a lipid transfer protein which is constitutively at least 50-fold more abundant in plants carrying the resistant allele of Qfhs.ifa-5A. In addition to this candidate gene associated with Qfhs.ifa-5A, we identified a uridine diphosphate (UDP)-glycosyltransferase gene, designated TaUGT12887, exhibiting a positive difference in response to the pathogen in lines harbouring both QTLs relative to lines carrying only the Qfhs.ifa-5A resistance allele, suggesting Fhb1 dependence of this transcript. Yet, this dependence was observed only in the NIL with already higher basal resistance. The complete cDNA of TaUGT12887 was reconstituted from available wheat genomic sequences, and a synthetic recoded gene was expressed in a toxin-sensitive strain of Saccharomyces cerevisiae. This gene conferred deoxynivalenol resistance, albeit much weaker than that observed with the previously characterized barley HvUGT13248.


Subject(s)
Disease Resistance/genetics , Fusarium/physiology , Genetic Association Studies , Quantitative Trait Loci/genetics , Transcriptome/genetics , Triticum/genetics , Triticum/microbiology , Carrier Proteins/metabolism , Disease Resistance/immunology , Fusarium/drug effects , Gene Expression Regulation, Plant/drug effects , Genes, Plant/genetics , Physical Chromosome Mapping , Plant Diseases/genetics , Plant Diseases/immunology , Plant Diseases/virology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic/drug effects , Transcriptome/drug effects , Trichothecenes/pharmacology , Triticum/immunology
6.
Cell Mol Life Sci ; 67(19): 3299-311, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20461437

ABSTRACT

Sprouty2 is an important inhibitor of cell proliferation and signal transduction. In this study, we found a bimodal expression of Sprouty2 protein during cell cycle progression after exit from quiescence, whereas elevated Sprouty4 expression in the G1 phase stayed high throughout the rest of the cell cycle. Induction of the mitogen-activated protein kinase via activated Ras was crucial for increased Sprouty2 expression at the G0/G1 transition. Following the first peak, accelerated proteasomal protein degradation caused a transient attenuation of Sprouty2 abundance during late G1. Since the decline in its expression was abolished by dominant negative c-Cbl and the timely restricted interaction between Sprouty2 and c-Cbl disappeared at the second peak of Sprouty2 expression, we conclude that the second phase in the cell cycle-specific expression profile of Sprouty2 is solely dependent on ubiquitination by c-Cbl. Our results suggest that Sprouty2 abundance is the result of strictly coordinated activities of Ras and c-Cbl.


Subject(s)
Cell Cycle/genetics , Genes, ras/genetics , Cell Line , Cell Line, Tumor , Fibroblasts/metabolism , Humans , Lung/pathology
7.
Biol Chem ; 391(7): 813-21, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20482313

ABSTRACT

Sprouty (Spry) proteins are well-known negative regulators of receptor tyrosine kinase-mediated signalling. Their expression is controlled by mitogens, implying a negative feedback loop. Correspondingly, the different members of the family fulfil important roles during organogenesis by adjustment of growth factor-induced processes. In addition, Spry4, one member of this protein family, has been shown to regulate angiogenesis by inhibiting vascular endothelial cell growth factor-induced extracellular signalling-regulated kinase (ERK) activation. Because oxygen is an important regulator of angiogenesis, we investigated Spry4 expression patterns under hypoxic conditions. Our data demonstrate that both hypoxia and desferrioxamine (DFO) treatment increased Spry4 expression. Following iron depletion, elevated Spry4 levels were detected in several cell types independent of tissue origin, presence of mitogens, cell differentiation and malignancy. Evaluation of the underlying regulative mechanisms revealed that augmented transcription and increased mRNA stability enhance mRNA levels of Spry4 in response to DFO. This study unveils a growth factor-independent regulation mechanism of Spry4 expression. Because increased Spry4 levels are accompanied by disappearing ERK phosphorylation, Spry4 might be involved in the timely restriction of MAPK signals under hypoxic conditions, similar to its role in mitogen-regulated processes. However, the functional significance of the observed upregulation of Spry4 during iron depletion remains to be clarified.


Subject(s)
Hypoxia/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Nerve Tissue Proteins/genetics , RNA Stability/genetics , Transcription, Genetic/genetics , Cell Hypoxia/genetics , Cells, Cultured , Humans , Reverse Transcriptase Polymerase Chain Reaction
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