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1.
EMBO J ; 39(1): e101533, 2020 01 02.
Article in English | MEDLINE | ID: mdl-31701553

ABSTRACT

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 "master" enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB.


Subject(s)
Chromatin/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation/drug effects , Inflammation Mediators/metabolism , Interleukin-1alpha/pharmacology , MAP Kinase Kinase Kinases/metabolism , NF-kappa B/metabolism , Binding Sites , Cells, Cultured , Chemokines/metabolism , Chromatin/chemistry , Chromatin/genetics , HeLa Cells , Humans , MAP Kinase Kinase Kinases/genetics , NF-kappa B/genetics , Signal Transduction , Tumor Necrosis Factor-alpha/pharmacology
2.
Cancers (Basel) ; 11(8)2019 Aug 16.
Article in English | MEDLINE | ID: mdl-31426445

ABSTRACT

The frequently occurring heterogeneity of cancer cells and their functional interaction with immune cells in the tumor microenvironment raises the need to study signaling pathways at the single cell level with high precision, sensitivity, and spatial resolution. As aberrant NF-κB activity has been implicated in almost all steps of cancer development, we analyzed the dynamic regulation and activation status of the canonical NF-κB pathway in control and IL-1α-stimulated individual cells using proximity ligation assays (PLAs). These systematic experiments allowed the visualization of the dynamic dissociation and re-formation of endogenous p65/IκBα complexes and the nuclear translocation of NF-κB p50/p65 dimers. PLA combined with immunostaining for p65 or with NFKBIA single molecule mRNA-FISH facilitated the analysis of (i) further levels of the NF-κB pathway, (i) its functionality for downstream gene expression, and (iii) the heterogeneity of the NF-κB response in individual cells. PLA also revealed the interaction between NF-κB p65 and the P-body component DCP1a, a new p65 interactor that contributes to efficient p65 NF-κB nuclear translocation. In summary, these data show that PLA technology faithfully mirrored all aspects of dynamic NF-κB regulation, thus allowing molecular diagnostics of this key pathway at the single cell level which will be required for future precision medicine.

3.
Plant Methods ; 15: 151, 2019.
Article in English | MEDLINE | ID: mdl-31889976

ABSTRACT

BACKGROUND: Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA. RESULTS: A protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages. CONCLUSION: Here we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes.

4.
PLoS Pathog ; 13(3): e1006286, 2017 03.
Article in English | MEDLINE | ID: mdl-28355270

ABSTRACT

Coronavirus replication takes place in the host cell cytoplasm and triggers inflammatory gene expression by poorly characterized mechanisms. To obtain more insight into the signals and molecular events that coordinate global host responses in the nucleus of coronavirus-infected cells, first, transcriptome dynamics was studied in human coronavirus 229E (HCoV-229E)-infected A549 and HuH7 cells, respectively, revealing a core signature of upregulated genes in these cells. Compared to treatment with the prototypical inflammatory cytokine interleukin(IL)-1, HCoV-229E replication was found to attenuate the inducible activity of the transcription factor (TF) NF-κB and to restrict the nuclear concentration of NF-κB subunits by (i) an unusual mechanism involving partial degradation of IKKß, NEMO and IκBα and (ii) upregulation of TNFAIP3 (A20), although constitutive IKK activity and basal TNFAIP3 expression levels were shown to be required for efficient virus replication. Second, we characterized actively transcribed genomic regions and enhancers in HCoV-229E-infected cells and systematically correlated the genome-wide gene expression changes with the recruitment of Ser5-phosphorylated RNA polymerase II and prototypical histone modifications (H3K9ac, H3K36ac, H4K5ac, H3K27ac, H3K4me1). The data revealed that, in HCoV-infected (but not IL-1-treated) cells, an extensive set of genes was activated without inducible p65 NF-κB being recruited. Furthermore, both HCoV-229E replication and IL-1 were shown to upregulate a small set of genes encoding immunomodulatory factors that bind p65 at promoters and require IKKß activity and p65 for expression. Also, HCoV-229E and IL-1 activated a common set of 440 p65-bound enhancers that differed from another 992 HCoV-229E-specific enhancer regions by distinct TF-binding motif combinations. Taken together, the study shows that cytoplasmic RNA viruses fine-tune NF-κB signaling at multiple levels and profoundly reprogram the host cellular chromatin landscape, thereby orchestrating the timely coordinated expression of genes involved in multiple signaling, immunoregulatory and metabolic processes.


Subject(s)
Chromatin/physiology , Coronavirus 229E, Human , Coronavirus Infections/genetics , NF-kappa B/metabolism , Transcriptome , Cell Line , Chromatin Immunoprecipitation , Gene Expression Regulation , Humans , Immunoblotting , Laser Capture Microdissection , Microscopy, Fluorescence , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction
6.
Mol Cell ; 62(6): 943-957, 2016 06 16.
Article in English | MEDLINE | ID: mdl-27315556

ABSTRACT

Signals and posttranslational modifications regulating the decapping step in mRNA degradation pathways are poorly defined. In this study we reveal the importance of K63-linked ubiquitylation for the assembly of decapping factors, P-body formation, and constitutive decay of instable mRNAs encoding mediators of inflammation by various experimental approaches. K63-branched ubiquitin chains also regulate IL-1-inducible phosphorylation of the P-body component DCP1a. The E3 ligase TRAF6 binds to DCP1a and indirectly regulates DCP1a phosphorylation, expression of decapping factors, and gene-specific mRNA decay. Mutation of six C-terminal lysines of DCP1a suppresses decapping activity and impairs the interaction with the mRNA decay factors DCP2, EDC4, and XRN1, but not EDC3, thus remodeling P-body architecture. The usage of ubiquitin chains for the proper assembly and function of the decay-competent mammalian decapping complex suggests an additional layer of control to allow a coordinated function of decapping activities and mRNA metabolism in higher eukaryotes.


Subject(s)
Endoribonucleases/metabolism , Lysine/metabolism , RNA Caps/metabolism , RNA Stability , RNA, Messenger/metabolism , TNF Receptor-Associated Factor 6/metabolism , Trans-Activators/metabolism , Ubiquitination , Animals , Cell Line, Tumor , Endoribonucleases/genetics , Exoribonucleases/metabolism , HEK293 Cells , Humans , Interleukin-1alpha/pharmacology , Intracellular Signaling Peptides and Proteins , Mice , Microtubule-Associated Proteins/metabolism , Mutation , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Proteins/metabolism , RNA Caps/genetics , RNA Stability/drug effects , RNA, Messenger/genetics , Receptors, Interleukin-1/agonists , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/metabolism , TNF Receptor-Associated Factor 6/genetics , Time Factors , Trans-Activators/genetics , Transfection , Ubiquitination/drug effects
7.
Mol Cell ; 53(2): 193-208, 2014 Jan 23.
Article in English | MEDLINE | ID: mdl-24389100

ABSTRACT

Given the intimate link between inflammation and dysregulated cell proliferation in cancer, we investigated cytokine-triggered gene expression in different cell cycle stages. Transcriptome analysis revealed that G1 release through cyclin-dependent kinase 6 (CDK6) and CDK4 primes and cooperates with the cytokine-driven gene response. CDK6 physically and functionally interacts with the NF-κB subunit p65 in the nucleus and is found at promoters of many transcriptionally active NF-κB target genes. CDK6 recruitment to distinct chromatin regions of inflammatory genes was essential for proper loading of p65 to its cognate binding sites and for the function of p65 coactivators, such as TRIP6. Furthermore, cytokine-inducible nuclear translocation and chromatin association of CDK6 depends on the kinase activity of TAK1 and p38. These results have widespread biological implications, as aberrant CDK6 expression or activation that is frequently observed in human tumors modulates NF-κB to shape the cytokine and chemokine repertoires in chronic inflammation and cancer.


Subject(s)
Chromatin/metabolism , Cyclin-Dependent Kinase 6/physiology , NF-kappa B/genetics , Cell Cycle/genetics , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/physiology , Cyclin-Dependent Kinase 6/analysis , Cyclin-Dependent Kinase 6/metabolism , Gene Expression Regulation , HeLa Cells , Humans , Interleukin-1/metabolism , Interleukin-1/physiology , Interleukin-8/genetics , Interleukin-8/metabolism , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Kinase Kinases/physiology , Promoter Regions, Genetic , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Transcription Factor RelA/physiology , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/physiology , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/physiology
8.
PLoS One ; 7(12): e51847, 2012.
Article in English | MEDLINE | ID: mdl-23300567

ABSTRACT

Nuclear factor kappa-B (NF-κB) activates multiple genes with overlapping roles in cell proliferation, inflammation and cancer. Using an unbiased approach we identified human CDK6 as a novel kinase phosphorylating NF-κB p65 at serine 536. Purified and reconstituted CDK6/cyclin complexes phosphorylated p65 in vitro and in transfected cells. The physiological role of CDK6 for basal as well as cytokine-induced p65 phosphorylation or NF-κB activation was revealed upon RNAi-mediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-κB and TNF-induced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-κB p65-mediated transcription in a target gene specific manner and this effect was partially dependent on its ability to phosphorylate p65 at serine 536. Tumor formation in thymi and spleens of v-cyclin transgenic mice correlated with increased levels of p65 Ser536 phosphorylation, increased expression of CDK6 and upregulaton of the NF-κB target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-κB to chronic inflammation and neoplasia.


Subject(s)
Cyclin-Dependent Kinase 6/metabolism , Gene Expression Regulation, Neoplastic , Inflammation/genetics , NF-kappa B/metabolism , Serine/metabolism , Splenic Neoplasms/genetics , Thymus Neoplasms/genetics , Animals , Apoptosis , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cell Proliferation , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/genetics , HeLa Cells , Humans , I-kappa B Proteins/metabolism , Immunoblotting , Inflammation/metabolism , Inflammation/pathology , Lymphocytes/metabolism , Lymphocytes/pathology , Mice , Mice, Transgenic , NF-kappa B/genetics , Phosphorylation , RNA, Small Interfering/genetics , Serine/chemistry , Serine/genetics , Splenic Neoplasms/metabolism , Splenic Neoplasms/pathology , Tandem Mass Spectrometry , Thymus Neoplasms/metabolism , Thymus Neoplasms/pathology , Tumor Necrosis Factor-alpha/pharmacology
9.
J Cell Biol ; 194(4): 581-96, 2011 Aug 22.
Article in English | MEDLINE | ID: mdl-21859862

ABSTRACT

Cytokines and stress-inducing stimuli signal through c-Jun N-terminal kinase (JNK) using a diverse and only partially defined set of downstream effectors. In this paper, the decapping complex subunit DCP1a was identified as a novel JNK target. JNK phosphorylated DCP1a at residue S315 in vivo and in vitro and coimmunoprecipitated and colocalized with DCP1a in processing bodies (P bodies). Sustained JNK activation by several different inducers led to DCP1a dispersion from P bodies, whereas IL-1 treatment transiently increased P body number. Inhibition of TAK1-JNK signaling also affected the number and size of P bodies and the localization of DCP1a, Xrn1, and Edc4. Transcriptome analysis further identified a central role for DCP1a in IL-1-induced messenger ribonucleic acid (mRNA) expression. Phosphomimetic mutation of S315 stabilized IL-8 but not IκBα mRNA, whereas overexpressed DCP1a blocked IL-8 transcription and suppressed p65 NF-κB nuclear activity. Collectively, these data reveal DCP1a as a multifunctional regulator of mRNA expression and suggest a novel mechanism controlling the subcellular localization of DCP1a in response to stress or inflammatory stimuli.


Subject(s)
Cytoplasmic Granules/enzymology , Endoribonucleases/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Trans-Activators/metabolism , Animals , Endoribonucleases/genetics , Enzyme Activation , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Expression Regulation , Genes, Reporter , HEK293 Cells , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Inflammation Mediators/metabolism , Interleukin-1/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , JNK Mitogen-Activated Protein Kinases/deficiency , JNK Mitogen-Activated Protein Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , Mice , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Phosphorylation , Protein Transport , Proteins/genetics , Proteins/metabolism , RNA Stability , RNA, Messenger/metabolism , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/metabolism , Recombinant Fusion Proteins/metabolism , Stress, Physiological , Time Factors , Trans-Activators/genetics , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Transfection
10.
PLoS One ; 6(12): e29256, 2011.
Article in English | MEDLINE | ID: mdl-22216226

ABSTRACT

TAB1 was defined as a regulatory subunit of the protein kinase TAK1, which functions upstream in the pathways activated by interleukin (IL)-1, tumor necrosis factor (TNF), toll-like receptors (TLRs) and stressors. However, TAB1 also functions in the p38 MAPK pathway downstream of TAK1. We identified amino acids (aa) 452/453 and 456/457 of TAB1 as novel sites phosphorylated by TAK1 as well as by p38 MAPK in intact cells as well as in vitro. Serines 452/453 and 456/457 were phosphorylated upon phosphatase blockade by calyculin A, or in response to IL-1 or translational stressors such as anisomycin and sorbitol. Deletion or phospho-mimetic mutations of aa 452-457 of TAB1 retain TAB1 and p38 MAPK in the cytoplasm. The TAB1 mutant lacking aa 452-457 decreases TAB1-dependent phosphorylation of p38 MAPK. It also enhances TAB1-dependent CCL5 secretion in response to IL-1 and increases activity of a post-transcriptional reporter gene, which contains the CCL5 3' untranslated region. These data suggest a complex role of aa 452-457 of TAB1 in controlling p38 MAPK activity and subcellular localization and implicate these residues in TAK1- or p38 MAPK-dependent post-transcriptional control of gene expression.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Amino Acid Sequence , Base Sequence , Cell Line , Cytokines/metabolism , Cytoplasm/metabolism , DNA Primers , Enzyme-Linked Immunosorbent Assay , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Molecular Sequence Data , Mutation , Phosphorylation , RNA Processing, Post-Transcriptional , Sequence Homology, Amino Acid , Stress, Physiological , Ubiquitination , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
11.
Hypertension ; 56(3): 490-7, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20606112

ABSTRACT

Arterial hypertension is associated with increased levels of reactive oxygen species, which may scavenge endothelium-derived NO and thereby diminish its vasorelaxant effects. However, the quantitatively relevant source of reactive oxygen species is unclear. Thus, this potential pathomechanism is not yet pharmacologically targetable. Several enzymatic sources of reactive oxygen species have been suggested: uncoupled endothelial NO synthase, xanthine oxidase, and NADPH oxidases. Here we show that increased reactive oxygen species formation in aortas of 12- to 14-month-old spontaneously hypertensive rats versus age-matched Wistar Kyoto rats is inhibited by the specific NADPH oxidase inhibitor VAS2870 but neither by the xanthine oxidase inhibitor oxypurinol nor the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester. NADPH oxidase activity, as well as protein expression of its catalytic subunits, NOX1 and NOX2, was increased in the aortas of spontaneously hypertensive rats, whereas the expression of NOX4 protein, the most abundant NOX isoform, was not significantly changed. Impaired acetylcholine-induced relaxation of spontaneously hypertensive rat aortas was significantly improved by VAS2870. In conclusion, NOX1 and NOX2 but not NOX4 proteins are increased in aged spontaneously hypertensive rat aortas. Importantly, these NOX isoforms, in particular, ectopic expression of NOX1 in endothelial cells, appear to affect vascular function in an NADPH oxidase inhibitor-reversible manner. NADPH oxidases may, thus, be a novel target for the treatment of systemic hypertension.


Subject(s)
Aorta/physiopathology , Endothelium, Vascular/physiopathology , Hypertension/physiopathology , Membrane Glycoproteins/metabolism , NADH, NADPH Oxidoreductases/metabolism , NADPH Oxidases/metabolism , Oxidative Stress/physiology , Acetylcholine/pharmacology , Aging , Analysis of Variance , Animals , Aorta/drug effects , Aorta/metabolism , Blotting, Western , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Fluorescent Antibody Technique , Hypertension/metabolism , Male , NADPH Oxidase 1 , NADPH Oxidase 2 , Nitric Oxide Synthase Type III/metabolism , Oxidative Stress/drug effects , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Reactive Oxygen Species/metabolism , Xanthine Oxidase/metabolism
12.
Exp Parasitol ; 126(4): 592-602, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20599986

ABSTRACT

Comparative analyses of the N-glycosylation pattern of hemolymph glycoproteins from Biomphalaria glabrata strains Puerto Rico (BgPR) and Salvador (BgBS-90), differing in their susceptibility towards Schistosoma mansoni infection, were performed by Western blotting, enzyme-linked immunosorbent assays, two-dimensional high-performance liquid chromatography and mass spectrometry. Obtained data demonstrated an enhanced expression of serologically cross-reacting, fucosylated carbohydrate epitopes by the highly susceptible BgPR-strain in comparison to the resistant BgBS-90-strain. In particular, glycoproteins of BgPR snails exhibited larger amounts of glycans with (ß1-2)-linked xylose or terminal Fuc(α1-3)GalNAc(ß1-4)[±Fuc(α1-3)]GlcNAc(ß1-)-units which are known to mediate cross-reactivity with schistosomal glycoconjugates. This finding could be corroborated by immunohistochemical studies showing again an enhanced expression of such carbohydrate epitopes in BgPR tissue. Hence, our results provide evidence for a correlation of B. glabrata susceptibility towards S. mansoni infection and the expression of carbohydrate determinants shared by the parasite and its intermediate host.


Subject(s)
Biomphalaria/chemistry , Glycoproteins/metabolism , Hemolymph/chemistry , Schistosoma mansoni/physiology , Animals , Antigens, Helminth/analysis , Antigens, Helminth/immunology , Biomphalaria/immunology , Biomphalaria/parasitology , Blotting, Western , Bulinus/chemistry , Carbohydrates/analysis , Carbohydrates/immunology , Chromatography, High Pressure Liquid , Cross Reactions , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Epitopes/analysis , Epitopes/immunology , Glycoproteins/immunology , Glycosylation , Immunohistochemistry , Polysaccharides/immunology , Polysaccharides/metabolism , Schistosoma mansoni/immunology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
13.
Toxins (Basel) ; 2(6): 1279-99, 2010 06.
Article in English | MEDLINE | ID: mdl-22069638

ABSTRACT

The release of tumor necrosis factor α (TNF-α) by ochratoxin A (OTA) was studied in various macrophage and non-macrophage cell lines and compared with E. coli lipopolysaccharide (LPS) as a standard TNF-α release agent. Cells were exposed either to 0, 2.5 or 12.5 µmol/L OTA, or to 0.1 µg/mL LPS, for up to 24 h. OTA at 2.5 µmol/L and LPS at 0.1 µg/mL were not toxic to the tested cells as indicated by viability markers. TNF-α was detected in the incubated cell medium of rat Kupffer cells, peritoneal rat macrophages, and the mouse monocyte macrophage cell line J774A.1: TNF-α concentrations were 1,000 pg/mL, 1,560 pg/mL, and 650 pg/mL, respectively, for 2.5 µmol/L OTA exposure and 3,000 pg/mL, 2,600 pg/mL, and 2,115 pg/mL, respectively, for LPS exposure. Rat liver sinusoidal endothelial cells, rat hepatocytes, human HepG2 cells, and mouse L929 cells lacked any cytokine response to OTA, but showed a significant release of TNF-α after LPS exposure, with the exception of HepG2 cells. In non-responsive cell lines, OTA lacked both any activation of NF-κB or the translocation of activated NF-κB to the cell nucleus, i.e., in mouse L929 cells. In J774A.1 cells, OTA mediated TNF-α release via the pRaf/MEK 1/2-NF-κB and p38-NF-κB pathways, whereas LPS used pRaf/MEK 1/2-NF-κB, but not p38-NF-κB pathways. In contrast, in L929 cells, LPS used other pathways to activate NF-κB. Our data indicate that only macrophages and macrophage derived cells respond to OTA and are considered as sources for TNF-α release upon OTA exposure.


Subject(s)
Lipopolysaccharides/toxicity , Ochratoxins/toxicity , Tumor Necrosis Factor-alpha/metabolism , Animals , Cell Line , Cell Survival/drug effects , Cells, Cultured , Drug Interactions , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Hep G2 Cells , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Rats , Rats, Wistar
14.
Eur J Cell Biol ; 86(8): 445-60, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17628207

ABSTRACT

The 'Solute Carrier Family SLC10' consists of six annotated members in humans, comprising two bile acid carriers (SLC10A1 and SLC10A2), one steroid sulfate transporter (SLC10A6), and three orphan carriers (SLC10A3 to SLC10A5). In this study we report molecular characterization and expression analysis of a novel member of the SLC10 family, SLC10A7, previously known as C4orf13. SLC10A7 proteins consist of 340-343 amino acids in humans, mice, rats, and frogs and show an overall amino acid sequence identity of >85%. SLC10A7 genes comprise 12 coding exons and show broad tissue expression pattern. When expressed in Xenopus laevis oocytes and HEK293 cells, SLC10A7 was detected in the plasma membrane but revealed no transport activity for bile acids and steroid sulfates. By immunofluorescence analysis of dual hemagglutinin (HA)- and FLAG-labeled SLC10A7 proteins in HEK293 cells, we established a topology of 10 transmembrane domains with an intracellular cis orientation of the N-terminal and C-terminal ends. This topology pattern is clearly different from the seven-transmembrane domain topology of the other SLC10 members but similar to hitherto uncharacterized non-vertebrate SLC10A7-related proteins. In contrast to the established SLC10 members, which are restricted to the taxonomic branch of vertebrates, SLC10A7-related proteins exist also in yeasts, plants, and bacteria, making SLC10A7 taxonomically the most widespread member of this carrier family. Vertebrate and bacterial SLC10A7 proteins exhibit >20% sequence identity, which is higher than the sequence identity of SLC10A7 to any other member of the SLC10 carrier family.


Subject(s)
Bacteria/genetics , Conserved Sequence , Organic Anion Transporters, Sodium-Dependent/genetics , Phylogeny , Vertebrates/genetics , Alternative Splicing/genetics , Amino Acid Sequence , Animals , Biological Transport , Cell Membrane/metabolism , Cloning, Molecular , Exons/genetics , Genome , Humans , Immunoprecipitation , Mice , Molecular Sequence Data , Oocytes , Organic Anion Transporters, Sodium-Dependent/chemistry , Organic Anion Transporters, Sodium-Dependent/metabolism , Rats , Recombinant Fusion Proteins/metabolism , Symporters/metabolism , Xenopus
15.
Proc Natl Acad Sci U S A ; 103(12): 4735-40, 2006 Mar 21.
Article in English | MEDLINE | ID: mdl-16537417

ABSTRACT

Natriuretic peptides (NP) mediate their effects by activating membrane-bound guanylyl cyclase-coupled receptors A (NPR-A) or B (NPR-B). Whereas the pathophysiological role of NPR-A has been widely studied, only limited knowledge on the cardiovascular function of NPR-B is available. In vitro studies suggest antiproliferative and antihypertrophic actions of the NPR-B ligand C-type NP (CNP). Because of the lack of a specific pharmacological inhibitor, these effects could not clearly be attributed to impaired NPR-B signaling. Recently, gene deletion revealed a predominant role of NPR-B in endochondral ossification and development of female reproductive organs. However, morphological abnormalities and premature death of NPR-B-deficient mice preclude detailed cardiovascular phenotyping. In the present study, a dominant-negative mutant (NPR-BDeltaKC) was used to characterize CNP-dependent NPR-B signaling in vitro and in transgenic rats. Here we demonstrate that reduced CNP- but not atrial NP-dependent cGMP response attenuates antihypertrophic potency of CNP in vitro. In transgenic rats, NPR-BDeltaKC expression selectively reduced NPR-B but not NPR-A signaling. NPR-BDeltaKC transgenic rats display progressive, blood pressure-independent cardiac hypertrophy and elevated heart rate. The hypertrophic phenotype is further enhanced in chronic volume overload-induced congestive heart failure. Thus, this study provides evidence linking NPR-B signaling to the control of cardiac growth.


Subject(s)
Genes, Dominant , Guanylate Cyclase/genetics , Hypertrophy, Left Ventricular/physiopathology , Receptors, Atrial Natriuretic Factor/genetics , Animals , Animals, Genetically Modified , Blood Pressure/genetics , Bone Development/genetics , Cyclic GMP/metabolism , Heart Rate/genetics , Heart Ventricles/drug effects , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Hypertrophy, Left Ventricular/genetics , Hypertrophy, Left Ventricular/pathology , Kidney/physiology , Mutation , Natriuretic Peptide, C-Type/pharmacology , Rats , Sequence Deletion
16.
Circulation ; 113(2): 286-95, 2006 Jan 17.
Article in English | MEDLINE | ID: mdl-16391154

ABSTRACT

BACKGROUND: Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. Using wild-type and homozygous endothelial nitric oxide synthase (NOS3(-/-)) knockout mice with pulmonary hypertension induced by chronic hypoxia and rats with monocrotaline-induced pulmonary hypertension, we examined whether the soluble guanylate cyclase (sGC) stimulator Bay41-2272 or the sGC activator Bay58-2667 could reverse pulmonary vascular remodeling. METHODS AND RESULTS: Both Bay41-2272 and Bay58-2667 dose-dependently inhibited the pressor response of acute hypoxia in the isolated perfused lung system. When wild-type (NOS3(+/+)) or NOS3(-/-) mice were housed under 10% oxygen conditions for 21 or 35 days, both strains developed pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling, demonstrated by an increase in fully muscularized peripheral pulmonary arteries. Treatment of wild-type mice with the activator of sGC, Bay58-2667 (10 mg/kg per day), or the stimulator of sGC, Bay41-2272 (10 mg/kg per day), after full establishment of pulmonary hypertension from day 21 to day 35 significantly reduced pulmonary hypertension, right ventricular hypertrophy, and structural remodeling of the lung vasculature. In contrast, only minor efficacy of chronic sGC activator therapies was noted in NOS3(-/-) mice. In monocrotaline-injected rats with established severe pulmonary hypertension, both compounds significantly reversed hemodynamic and structural changes. CONCLUSIONS: Activation of sGC reverses hemodynamic and structural changes associated with monocrotaline- and chronic hypoxia-induced experimental pulmonary hypertension. This effect is partially dependent on endogenous nitric oxide generated by NOS3.


Subject(s)
Cardiomegaly/enzymology , Guanylate Cyclase/metabolism , Hypertension, Pulmonary/enzymology , Pulmonary Circulation , Animals , Benzoates/administration & dosage , Benzoates/pharmacology , Disease Models, Animal , Enzyme Activation/drug effects , Humans , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/etiology , Hypertrophy, Right Ventricular/enzymology , Hypoxia/complications , Male , Mice , Mice, Knockout , Nitric Oxide Synthase Type II/deficiency , Nitric Oxide Synthase Type III , Pyrazoles/administration & dosage , Pyrazoles/pharmacology , Pyridines/administration & dosage , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Solubility
17.
J Mol Histol ; 36(5): 345-53, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16240167

ABSTRACT

Biogenic amines (serotonin and catecholamines), play an important role in the control of the blood flow not only in vertebrates, but also in invertebrates such as cephalopods. In contrast to the well investigated hearts of the a 'modern', coleoid cephalopods, the innervation of the heart of the archaic Nautilus pompilius L. has not been studied in detail. In this study the distribution and effects of biogenic amines in the Nautilus heart were investigated. Serotonin and catecholamines were visualised by the glyxoylic acid induced fluorescence. High performance liquid chromatotography analysis was performed to discriminate between the catecholamines, which showed a high content of noradrenaline in the 4 auricles, the aorta and the ventricle, whereas the ventricle showed a high dopamine content. Adrenaline was found at a very low concentration in the ventricle. Serotonin and dopamine were also immunohistochemically localised to larger nerves and throughout the heart, respectively. In organ bath experiments, the auricles showed little spontaneous activity. After adding serotonin, they displayed rhythmical contractions, which were accelerated dose-dependently by noradrenaline. In summary, these data suggest an important role for biogenic amines in the control of the heart of Nautilus pompilius L., with serotonin possibly stimulating excitatory nerve fibres, whereas noradrenaline is likely to influence the muscle contraction itself.


Subject(s)
Biogenic Amines/metabolism , Cephalopoda/metabolism , Myocardium/metabolism , Animals , Biogenic Amines/physiology , Cephalopoda/enzymology , Chromatography, High Pressure Liquid , Dopamine/metabolism , Female , Fluorescence , Glyoxylates/pharmacology , Heart Ventricles/metabolism , Immunohistochemistry , Male , Monoamine Oxidase/metabolism , Nerve Fibers/metabolism , Pericardium/enzymology , Serotonin/metabolism , Tissue Distribution
18.
J Morphol ; 264(3): 277-85, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15803486

ABSTRACT

This study presents histological and cytological findings on the structural differentiation of the mantle of Nautilus pompilius in order to characterize the cells that are responsible for shell formation. The lateral and front mantle edges split distally into three folds: an outer, middle, and inner fold. Within the upper part of the mantle the mantle edge is divided into two folds only; the inner fold disappears where the hood is attached to the mantle. At the base of the outer fold of the lateral and front mantle edge an endo-epithelial gland, the mantle edge gland, is localized. The gland cells are distinguished by a distinct rough endoplasmic reticulum and by numerous secretory vesicles. Furthermore, they show a strong accumulation of calcium compounds, indicating that the formation of the shell takes place in this region of the mantle. Numerous synaptic contacts between the gland cells and the axons of the nerve fibers reveal that the secretion in the area of the mantle edge gland is under nervous control. The whole mantle tissue is covered with a columnar epithelium having a microvillar border. The analyses of the outer epithelium show ultrastructural characteristics of a transport active epithelium, indicating that this region of the mantle is involved in the sclerotization of the shell. Ultrastructural findings concerning the epithelium between the outer and middle fold suggest that the periostracum is formed in this area of the mantle, as it is in other conchiferan molluscs.


Subject(s)
Mollusca/anatomy & histology , Animals , Microscopy, Electron, Scanning , Mollusca/physiology
19.
J Exp Zool A Comp Exp Biol ; 301(12): 930-7, 2004 Dec 01.
Article in English | MEDLINE | ID: mdl-15562500

ABSTRACT

Observations on the growth rate of aquarium maintained Nautilus pompilius in different developmental stages, i.e. juveniles (shell length about 8.75 cm), late juveniles (approximately 10 cm), and early adolescent (approximately 13.5 cm), indicate that this species is fully grown at an age of 7.3-8 years. The age calculations are based on two different computations: (1) the measurement of the increase of the shell length per day and (2) the formation of new septa in time intervals of 150+/-5 days, as demonstrated by X-ray analyses. After N. pompilius hatches, its shell grows about 139 mm to reach full growth and approximately 28 septa are formed. With an increase of the shell length of 0.052 mm per day, it takes about 2,673 days (7.3 years) to reach maturity. Provided that the process of chamber formation follows an exponential function, these computations result in approximately 2,925 days (8 years) to reach full maturity. Supposing that N. pompilius may live for several years after onset of maturity like Nautilus belauensis, the total life span for this species may exceed 11-12 years.


Subject(s)
Aging/physiology , Mollusca/growth & development , Age Determination by Skeleton , Animals , Body Weights and Measures , Radiography
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