RESUMEN
Transcription and export (TREX) is a multi-subunit complex that links synthesis, processing and export of mRNAs. It interacts with the RNA helicase UAP56 and export factors such as MOS11 and ALYs to facilitate nucleocytosolic transport of mRNAs. Plant MOS11 is a conserved, but sparsely researched RNA-binding export factor, related to yeast Tho1 and mammalian CIP29/SARNP. Using biochemical approaches, the domains of Arabidopsis thaliana MOS11 required for interaction with UAP56 and RNA-binding were identified. Further analyses revealed marked genetic interactions between MOS11 and ALY genes. Cell fractionation in combination with transcript profiling demonstrated that MOS11 is required for export of a subset of mRNAs that are shorter and more GC-rich than MOS11-independent transcripts. The central α-helical domain of MOS11 proved essential for physical interaction with UAP56 and for RNA-binding. MOS11 is involved in the nucleocytosolic transport of mRNAs that are upregulated under stress conditions and accordingly mos11 mutant plants turned out to be sensitive to elevated NaCl concentrations and heat stress. Collectively, our analyses identify functional interaction domains of MOS11. In addition, the results establish that mRNA export is critically involved in the plant response to stress conditions and that MOS11 plays a prominent role at this.
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Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , ARN Mensajero , Proteínas de Unión al ARN , Estrés Fisiológico , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Estrés Fisiológico/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Unión Proteica , Mutación/genética , Transporte de ARN , Dominios ProteicosRESUMEN
Transcript elongation by RNA polymerase II (RNAPII) is dynamic and highly regulated, thereby contributing to the implementation of gene expression programs during plant development or in response to environmental cues. The heterohexameric polymerase-associated factor 1 complex (PAF1C) stabilizes the RNAPII elongation complex promoting efficient transcript synthesis. In addition, PAF1C links transcriptional elongation with various post-translational histone modifications at transcribed loci. We have exposed Arabidopsis mutants deficient in the PAF1C subunits ELF7 or CDC73 to elevated NaCl concentrations to provoke a transcriptional response. The growth of elf7 plants was reduced relative to that of wildtype under these challenging conditions, whereas cdc73 plants exhibited rather enhanced tolerance. Profiling of the transcriptional changes upon NaCl exposure revealed that cdc73 responded similar to wildtype. Relative to wildtype and cdc73, the transcriptional response of elf7 plants was severely reduced in accord with their greater susceptibility to NaCl. The data also imply that CDC73 is more relevant for the transcription of longer genes. Despite the fact that both ELF7 and CDC73 are part of PAF1C the strikingly different transcriptional response of the mutants upon NaCl exposure suggests that the subunits have (partially) specific functions.
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BACKGROUND: Dysfunctional humoral and cellular innate immunity are key components in the development and progression of age-related macular degeneration (AMD). Specifically, chronically activated microglia and their disturbed regulatory system contribute to retinal degeneration. Galectin-3, a ß-galactose binding protein, is a potent driver of macrophage and microglia activation and has been implicated in neuroinflammation, including neurodegenerative diseases of the brain. Here, we hypothesized that genetic deficiency of galectin-3 or its modulation via TD139 dampens mononuclear phagocyte reactivity and delays retinal degeneration. METHODS: Galectin-3 expression in AMD patients was analyzed by immunohistochemical stainings. Galectin-3 knockout and BALB/cJ mice were exposed to white bright light with an intensity of 15,000 lux for 1 h and Cx3cr1GFP/+ mice to focal blue light of 50,000 lux for 10 min. BALB/cJ and Cx3cr1GFP/+ mice received intraperitoneal injections of 15 mg/kg TD139 or vehicle for five consecutive days, starting one day prior to light exposure. The effects of galectin-3 deficiency or inhibition on microglia were analyzed by immunohistochemical stainings and in situ hybridization of retinal sections and flat mounts. Pro-inflammatory cytokine levels in the retina and retinal pigment epithelium (RPE) were quantified by qRT-PCR and transcriptomic changes were analyzed by RNA-sequencing. Retinal thickness and structure were evaluated by optical coherence tomography. RESULTS: We found that galectin-3 expression was strongly upregulated in reactive retinal mononuclear phagocytes of AMD patients and in the two related mouse models of light-induced retinal degeneration. The experimental in vivo data further showed that specific targeting of galectin-3 by genetic knockout or administration of the small-molecule inhibitor TD139 reduced microglia reactivity and delayed retinal damage in both light damage conditions. CONCLUSION: This study defines galectin-3 as a potent driver of retinal degeneration and highlights the protein as a drug target for ocular immunomodulatory therapies.
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Galectina 3 , Degeneración Macular , Microglía , Animales , Citocinas/metabolismo , Galectina 3/antagonistas & inhibidores , Galectina 3/genética , Galectina 3/metabolismo , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/prevención & control , Ratones , Microglía/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , ARN/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/prevención & control , Tiogalactósidos/farmacología , Triazoles/farmacologíaRESUMEN
Both the dental pulp and the apical papilla represent a promising source of mesenchymal stem cells for regenerative endodontic protocols. The aim of this study was to outline molecular biological conformities and differences between dental pulp stem cells (DPSC) and stem cells from the apical papilla (SCAP). Thus, cells were isolated from the pulp and the apical papilla of an extracted molar and analyzed for mesenchymal stem cell markers as well as multi-lineage differentiation. During induced osteogenic differentiation, viability, proliferation, and wound healing assays were performed, and secreted signaling molecules were quantified by enzyme-linked immunosorbent assays (ELISA). Transcriptome-wide gene expression was profiled by microarrays and validated by quantitative reverse transcription PCR (qRT-PCR). Gene regulation was evaluated in the context of culture parameters and functionality. Both cell types expressed mesenchymal stem cell markers and were able to enter various lineages. DPSC and SCAP showed no significant differences in cell viability, proliferation, or migration; however, variations were observed in the profile of secreted molecules. Transcriptome analysis revealed the most significant gene regulation during the differentiation period, and 13 biomarkers were identified whose regulation was essential for both cell types. DPSC and SCAP share many features and their differentiation follows similar patterns. From a molecular biological perspective, both seem to be equally suitable for dental pulp tissue engineering.
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Células Madre Mesenquimatosas , Osteogénesis , Diferenciación Celular/genética , Proliferación Celular , Células Cultivadas , Papila Dental , Pulpa Dental , Osteogénesis/genética , Células MadreRESUMEN
Objectives: Multipotent mesenchymal stromal cells (MSC) play a pivotal role in the bone marrow (BM) niche. Stanniocalcin 1 (STC1) secreted by MSC has been demonstrated to promote the survival of neoplastic cells and was suggested a marker for minimal residual disease of acute myeloid leukemia (AML). Therefore, we evaluated the expression of STC1 in MSC from AML patients (MSCAML) compared to MSC from healthy donors (MSCHD).Methods: Liquid culture assays of MSCAML and MSCHD were performed to compare expansion capacity. Gene expression profiles of MSCAML vs. MSCHD were established. Secretion of STC1 was tested by ELISA in MSCAML vs. MSCHD and expression of STC1 in AML- vs. HD-BM by immunohistochemistry. In addition, co-cultures of AML cells on MSC were initiated and ultrastructural intercellular communication patterns were investigated. Finally, the effect of blocking STC1 on AML cells was evaluated.Results: MSCAML showed significant decreased expansion capacity compared to MSCHD. Gene analysis revealed marked overexpression of STC1 in MSCAML. ELISA and immunohistochemical findings confirmed this observation. Electron microscopy analysis showed reciprocal stimulation between AML cells and MSC. Blockade of STC1 did not significantly affect AML cell proliferation and apoptosis.Discussion: Characteristics of MSC differ depending on whether they originate from AML patients or from HD. STC1 was mostly overexpressed in MSCAML compared to MSCHD. In vitro blockade of STC1, however, was not associated with AML cell proliferation and apoptosis.Conclusion: Differences in expression levels of glycoproteins from MSCAML compared to MSCHD not necessarily assume that these molecules are niche-relevant in leukemic disease.
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Glicoproteínas/genética , Leucemia Mieloide Aguda/genética , Células Madre Mesenquimatosas/patología , Regulación hacia Arriba , Adulto , Anciano , Células Cultivadas , Femenino , Glicoproteínas/análisis , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
Background: Although diabetic nephropathy (DN) is the most common cause for end-stage renal disease in western societies, its pathogenesis still remains largely unclear. A different gene pattern of diabetic and healthy kidney cells is one of the probable explanations. Numerous signalling pathways have emerged as important pathophysiological mechanisms for diabetes-induced renal injury. Methods: Glomerular cells, as podocytes or mesangial cells, are predominantly involved in the development of diabetic renal lesions. While many gene assays concerning DN are performed with whole kidney or renal cortex tissue, we isolated glomeruli from black and tan, brachyuric (BTBR) obese/obese (ob/ob) and wildtype mice at four different timepoints (4, 8, 16 and 24 weeks) and performed an mRNA microarray to identify differentially expressed genes (DEGs). In contrast to many other diabetic mouse models, these homozygous ob/ob leptin-deficient mice develop not only a severe type 2 diabetes, but also diabetic kidney injury with all the clinical and especially histologic features defining human DN. By functional enrichment analysis we were able to investigate biological processes and pathways enriched by the DEGs at different disease stages. Altered expression of nine randomly selected genes was confirmed by quantitative polymerase chain reaction from glomerular RNA. Results: Ob/ob type 2 diabetic mice showed up- and downregulation of genes primarily involved in metabolic processes and pathways, including glucose, lipid, fatty acid, retinol and amino acid metabolism. Members of the CYP4A and ApoB family were found among the top abundant genes. But more interestingly, altered gene loci showed enrichment for processes and pathways linked to angioneogenesis, complement cascades, semaphorin pathways, oxidation and reduction processes and renin secretion. Conclusion: The gene profile of BTBR ob/ob type 2 diabetic mice we conducted in this study can help to identify new key players in molecular pathogenesis of diabetic kidney injury.
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Diabetes Mellitus Experimental , Nefropatías Diabéticas/genética , Regulación de la Expresión Génica , Glomérulos Renales/metabolismo , ARN/genética , Animales , Nefropatías Diabéticas/metabolismo , Modelos Animales de Enfermedad , Glomérulos Renales/patología , Ratones , Ratones Endogámicos , Ratones Obesos , Podocitos/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Estrogen receptor (ER) ß has been suggested to affect ovarian carcinogenesis. We examined the effects of four ERß agonists on proliferation and gene expression of two ovarian cancer cell lines. METHODS: OVCAR-3 and OAW-42 ovarian cancer cells were treated with the ERß agonists ERB-041, WAY200070, Liquiritigenin and 3ß-Adiol and cell growth was measured by means of the Cell Titer Blue Assay (Promega). ERß expression was knocked down by transfection with specific siRNA. Additionally, transcriptome analyses were performed by means of Affymetrix GeneChip arrays. To confirm the results of DNA microarray analysis, Western blot experiments were performed. RESULTS: All ERß agonists tested significantly decreased proliferation of OVCAR-3 and OAW-42 cells at a concentration of 10 nM. Maximum antiproliferative effects were induced by flavonoid Liquiritigenin, which inhibited growth of OVCAR-3 cells by 31.2% after 5 days of treatment, and ERB-041 suppressing proliferation of the same cell line by 29.1%. In OAW-42 cells, maximum effects were observed after treatment with the ERß agonist WAY200070, inhibiting cell growth by 26.8%, whereas ERB-041 decreased proliferation by 24.4%. In turn, knockdown of ERß with specific siRNA increased cell growth of OAW-42 cells about 1.9-fold. Transcriptome analyses revealed a set of genes regulated by ERß agonists including ND6, LCN1 and PTCH2, providing possible molecular mechanisms underlying the observed antiproliferative effects. CONCLUSION: In conclusion, the observed growth-inhibitory effects of all ERß agonists on ovarian cancer cell lines in vitro encourage further studies to test their possible use in the clinical setting.
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Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Receptor beta de Estrógeno/agonistas , Estrógenos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Androstano-3,17-diol/farmacología , Androstano-3,17-diol/uso terapéutico , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Estrógenos/uso terapéutico , Femenino , Flavanonas/farmacología , Flavanonas/uso terapéutico , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/fisiopatología , Oxazoles/farmacología , Oxazoles/uso terapéutico , Fenoles/farmacología , Fenoles/uso terapéuticoRESUMEN
BACKGROUND: Estrogen receptor ß (ERß) is expressed in the majority of invasive breast cancer cases, irrespective of their subtype, including triple-negative breast cancer (TNBC). Thus, ERß might be a potential target for therapy of this challenging cancer type. In this in vitro study, we examined the role of ERß in invasion of two triple-negative breast cancer cell lines. METHODS: MDA-MB-231 and HS578T breast cancer cells were treated with the specific ERß agonists ERB-041, WAY200070, Liquiritigenin and 3ß-Adiol. Knockdown of ERß expression was performed by means of siRNA transfection. Effects on cellular invasion were assessed in vitro by means of a modified Boyden chamber assay. Transcriptome analyses were performed using Affymetrix Human Gene 1.0 ST microarrays. Pathway and gene network analyses were performed by means of Genomatix and Ingenuity Pathway Analysis software. RESULTS: Invasiveness of MBA-MB-231 and HS578T breast cancer cells decreased after treatment with ERß agonists ERB-041 and WAY200070. Agonists Liquiritigenin and 3ß-Adiol only reduced invasion of MDA-MB-231 cells. Knockdown of ERß expression increased invasiveness of MDA-MB-231 cells about 3-fold. Transcriptome and pathway analyses revealed that ERß knockdown led to activation of TGFß signalling and induced expression of a network of genes with functions in extracellular matrix, tumor cell invasion and vitamin D3 metabolism. CONCLUSIONS: Our data suggest that ERß suppresses invasiveness of triple-negative breast cancer cells in vitro. Whether ERß agonists might be useful drugs in the treatment of triple-negative breast cancer, has to be evaluated in further animal and clinical studies.
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Movimiento Celular/efectos de los fármacos , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/antagonistas & inhibidores , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Técnicas In Vitro , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcriptoma/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Microglia reactivity is a hallmark of retinal degenerations and overwhelming microglial responses contribute to photoreceptor death. Minocycline, a semi-synthetic tetracycline analog, has potent anti-inflammatory and neuroprotective effects. Here, we investigated how minocycline affects microglia in vitro and studied its immuno-modulatory properties in a mouse model of acute retinal degeneration using bright white light exposure. METHODS: LPS-treated BV-2 microglia were stimulated with 50 µg/ml minocycline for 6 or 24 h, respectively. Pro-inflammatory gene transcription was determined by real-time RT-PCR and nitric oxide (NO) secretion was assessed using the Griess reagent. Caspase 3/7 levels were determined in 661W photoreceptors cultured with microglia-conditioned medium in the absence or presence of minocycline supplementation. BALB/cJ mice received daily intraperitoneal injections of 45 mg/kg minocycline, starting 1 day before exposure to 15.000 lux white light for 1 hour. The effect of minocycline treatment on microglial reactivity was analyzed by immunohistochemical stainings of retinal sections and flat-mounts, and messenger RNA (mRNA) expression of microglia markers was determined using real-time RT-PCR and RNA-sequencing. Optical coherence tomography (OCT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings were used to measure the extent of retinal degeneration and photoreceptor apoptosis. RESULTS: Stimulation of LPS-activated BV-2 microglia with minocycline significantly diminished the transcription of the pro-inflammatory markers CCL2, IL6, and inducible nitric oxide synthase (iNOS). Minocycline also reduced the production of NO and dampened microglial neurotoxicity on 661W photoreceptors. Furthermore, minocycline had direct protective effects on 661W photoreceptors by decreasing caspase 3/7 activity. In mice challenged with white light, injections of minocycline strongly decreased the number of amoeboid alerted microglia in the outer retina and down-regulated the expression of the microglial activation marker translocator protein (18 kDa) (TSPO), CD68, and activated microglia/macrophage whey acidic protein (AMWAP) already 1 day after light exposure. Furthermore, RNA-seq analyses revealed the potential of minocycline to globally counter-regulate pro-inflammatory gene transcription in the light-damaged retina. The severe thinning of the outer retina and the strong induction of photoreceptor apoptosis induced by light challenge were nearly completely prevented by minocycline treatment as indicated by a preserved retinal structure and a low number of apoptotic cells. CONCLUSIONS: Minocycline potently counter-regulates microgliosis and light-induced retinal damage, indicating a promising concept for the treatment of retinal pathologies.
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Antiinflamatorios/farmacología , Microglía/efectos de los fármacos , Microglía/patología , Minociclina/farmacología , Fármacos Neuroprotectores/farmacología , Retina/patología , Degeneración Retiniana/tratamiento farmacológico , Animales , Caspasas/metabolismo , Mediadores de Inflamación/metabolismo , Luz/efectos adversos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Degeneración Nerviosa/prevención & control , Óxido Nítrico/metabolismo , Degeneración Retiniana/patología , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/patologíaRESUMEN
BACKGROUND: Microglial cells are important effectors of the neuronal innate immune system with a major role in chronic neurodegenerative diseases. Curcumin, a major component of tumeric, alleviates pro-inflammatory activities of these cells by inhibiting nuclear factor kappa B (NFkB) signaling. To study the immuno-modulatory effects of curcumin on a transcriptomic level, DNA-microarray analyses were performed with resting and LPS-challenged microglial cells after short-term treatment with curcumin. METHODS: Resting and LPS-activated BV-2 cells were stimulated with curcumin and genome-wide mRNA expression patterns were determined using DNA-microarrays. Selected qRT-PCR analyses were performed to confirm newly identified curcumin-regulated genes. The migration potential of microglial cells was determined with wound healing assays and transwell migration assays. Microglial neurotoxicity was estimated by morphological analyses and quantification of caspase 3/7 levels in 661W photoreceptors cultured in the presence of microglia-conditioned medium. RESULTS: Curcumin treatment markedly changed the microglial transcriptome with 49 differentially expressed transcripts in a combined analysis of resting and activated microglial cells. Curcumin effectively triggered anti-inflammatory signals as shown by induced expression of Interleukin 4 and Peroxisome proliferator activated receptor α. Several novel curcumin-induced genes including Netrin G1, Delta-like 1, Platelet endothelial cell adhesion molecule 1, and Plasma cell endoplasmic reticulum protein 1, have been previously associated with adhesion and cell migration. Consequently, curcumin treatment significantly inhibited basal and activation-induced migration of BV-2 microglia. Curcumin also potently blocked gene expression related to pro-inflammatory activation of resting cells including Toll-like receptor 2 and Prostaglandin-endoperoxide synthase 2. Moreover, transcription of NO synthase 2 and Signal transducer and activator of transcription 1 was reduced in LPS-triggered microglia. These transcriptional changes in curcumin-treated LPS-primed microglia also lead to decreased neurotoxicity with reduced apoptosis of 661W photoreceptor cultures. CONCLUSIONS: Collectively, our results suggest that curcumin is a potent modulator of the microglial transcriptome. Curcumin attenuates microglial migration and triggers a phenotype with anti-inflammatory and neuroprotective properties. Thus, curcumin could be a nutraceutical compound to develop immuno-modulatory and neuroprotective therapies for the treatment of various neurodegenerative disorders.
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Antiinflamatorios no Esteroideos/farmacología , Movimiento Celular/efectos de los fármacos , Curcumina/farmacología , Expresión Génica/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/fisiología , Animales , Línea Celular , Perfilación de la Expresión Génica , Lipopolisacáridos/farmacología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcriptoma/efectos de los fármacosRESUMEN
BACKGROUND: Luteolin, a plant derived flavonoid, exerts a variety of pharmacological activities and anti-oxidant properties associated with its capacity to scavenge oxygen and nitrogen species. Luteolin also shows potent anti-inflammatory activities by inhibiting nuclear factor kappa B (NFkB) signaling in immune cells. To better understand the immuno-modulatory effects of this important flavonoid, we performed a genome-wide expression analysis in pro-inflammatory challenged microglia treated with luteolin and conducted a phenotypic and functional characterization. METHODS: Resting and LPS-activated BV-2 microglia were treated with luteolin in various concentrations and mRNA levels of pro-inflammatory markers were determined. DNA microarray experiments and bioinformatic data mining were performed to capture global transcriptomic changes following luteolin stimulation of microglia. Extensive qRT-PCR analyses were carried out for an independent confirmation of newly identified luteolin-regulated transcripts. The activation state of luteolin-treated microglia was assessed by morphological characterization. Microglia-mediated neurotoxicity was assessed by quantifying secreted nitric oxide levels and apoptosis of 661W photoreceptors cultured in microglia-conditioned medium. RESULTS: Luteolin dose-dependently suppressed pro-inflammatory marker expression in LPS-activated microglia and triggered global changes in the microglial transcriptome with more than 50 differentially expressed transcripts. Pro-inflammatory and pro-apoptotic gene expression was effectively blocked by luteolin. In contrast, mRNA levels of genes related to anti-oxidant metabolism, phagocytic uptake, ramification, and chemotaxis were significantly induced. Luteolin treatment had a major effect on microglial morphology leading to ramification of formerly amoeboid cells associated with the formation of long filopodia. When co-incubated with luteolin, LPS-activated microglia showed strongly reduced NO secretion and significantly decreased neurotoxicity on 661W photoreceptor cultures. CONCLUSIONS: Our findings confirm the inhibitory effects of luteolin on pro-inflammatory cytokine expression in microglia. Moreover, our transcriptomic data suggest that this flavonoid is a potent modulator of microglial activation and affects several signaling pathways leading to a unique phenotype with anti-inflammatory, anti-oxidative, and neuroprotective characteristics. With the identification of several novel luteolin-regulated genes, our findings provide a molecular basis to understand the versatile effects of luteolin on microglial homeostasis. The data also suggest that luteolin could be a promising candidate to develop immuno-modulatory and neuroprotective therapies for the treatment of neurodegenerative disorders.
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Antiinflamatorios/farmacología , Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Luteolina/farmacología , Microglía/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Células Cultivadas , Biología Computacional/métodos , Medios de Cultivo Condicionados/farmacología , Citocinas/genética , Perfilación de la Expresión Génica/métodos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Microglía/química , Óxido Nítrico/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Transducción de Señal/efectos de los fármacosRESUMEN
The human mitochondrial genome consists of a multicopy, circular dsDNA molecule of 16,569 base pairs. It encodes for 13 proteins, two ribosomal genes, and 22 tRNAs that are essential in the generation of cellular ATP by oxidative phosphorylation in eukaryotic cells. Germline mutations in mitochondrial DNA (mtDNA) are an important cause of maternally inherited diseases, while somatic mtDNA mutations may play important roles in aging and cancer. mtDNA polymorphisms are also widely used in population and forensic genetics. Therefore, methods that allow the rapid, inexpensive and accurate sequencing of mtDNA are of great interest. One such method is the Affymetrix GeneChip Human Mitochondrial Resequencing Array 2.0 (MitoChip v.2.0) (Santa Clara, CA). A direct comparison of 93 worldwide mitochondrial genomes sequenced by both the MitoChip and dideoxy terminator sequencing revealed an average call rate of 99.48% and an accuracy of > or =99.98% for the MitoChip. The good performance was achieved by using in-house software for the automated analysis of additional probes on the array that cover the most common haplotypes in the hypervariable regions (HVR). Failure to call a base was associated mostly with the presence of either a run of > or =4 C bases or a sequence variant within 12 bases up- or downstream of that base. A major drawback of the MitoChip is its inability to detect insertions/deletions and its low sensitivity and specificity in the detection of heteroplasmy. However, the vast majority of haplogroup defining polymorphism in the mtDNA phylogeny could be called unambiguously and more rapidly than with conventional sequencing.
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ADN Mitocondrial/química , Genoma Mitocondrial , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Genoma Humano , Humanos , Análisis de Secuencia de ADNRESUMEN
Although ChIP-chip is a powerful tool for genome-wide discovery of transcription factor target genes, the steps involving raw data analysis, identification of promoters, and correlation with binding sites are still laborious processes. Therefore, we report an integrated workflow for the analysis of promoter tiling arrays with the Genomatix ChipInspector system. We compare this tool with open-source software packages to identify PU.1 regulated genes in mouse macrophages. Our results suggest that ChipInspector data analysis, comparative genomics for binding site prediction, and pathway/network modeling significantly facilitate and enhance whole-genome promoter profiling to reveal in vivo sites of transcription factor-DNA interactions.
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Inmunoprecipitación de Cromatina , Análisis de Secuencia por Matrices de Oligonucleótidos , Estadística como Asunto , Animales , Sitios de Unión , Ratones , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismoRESUMEN
Sphingolipids are important components of the water permeability barrier of the skin. Moreover, ceramides were also shown to influence keratinocyte differentiation and regulate cellular signalling. A confluence-induced differentiation model of normal human keratinocytes was established to allow evaluation of pro- and anti-differentiation effects of exogenous compounds. The effects of phytosphingosine (PS), sphingosine (SO), sphinganine (SA) and their hexanoyl (-C6), stearoyl (-C18) and salicyl (-SLC) derivatives, C12-alkylamine-salicylate (C12-SLC), salicylate (SLC) along with vitamin D3 (VD3) and retinol as control substances were tested in this system. Cytotoxicity assays were carried out to optimize the incubation conditions of compounds and whole genome expression changes were monitored by DNA-microarray on days 0, 1 and 4. Geometric means of gene expression levels of a subset of known keratinocyte differentiation-related genes were calculated from the microarray data to compare effects of the sphingolipid derivatives. Compound treatment-induced transcriptional changes were analysed by the ExPlain software (BIOBASE GmbH). Five of the assayed substances (SA, SO-C6, PS-C6, SO-SLC, PS-SLC) were found to be potent promoters of keratinocyte differentiation compared with VD3, and C12-SLC revealed potential anti-differentiation properties. ExPlain analysis found a different regulatory profile in the computed transcriptional networks of the sphingoid bases versus their -C6 and especially -SLC derivatives suggesting that the change in their keratinocyte differentiation modifying potential is due to a unique effect of the covalent attachment of the salicylic acid. Taken together, these results demonstrate the gene regulatory potential of sphingolipid species that could be valuable for dermatological or cosmetic applications.
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Diferenciación Celular/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Esfingolípidos/farmacología , Adulto , Antígenos de Diferenciación/genética , Secuencia de Bases , Sitios de Unión , Diferenciación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Colecalciferol/farmacología , Femenino , Proteínas Filagrina , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular , Proteínas de Filamentos Intermediarios/genética , Queratina-10/genética , Queratinocitos/citología , Queratinocitos/metabolismo , Persona de Mediana Edad , Modelos Genéticos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Salicilatos/farmacología , Transglutaminasas/genética , Vitamina A/farmacologíaRESUMEN
A disaccharide degradation product of chondrotin sulfate proteoglycan-disaccharide (CSPG-DS) has been implicated previously in the inhibition of neurodegeneration by influencing microglia activation. In this study, genome-wide microarray analysis was used to identify specific gene expression profiles of CSPG-DS-stimulated BV-2 microglia-like cells. Gene products involved in phagocytosis, detoxification, migration, immune regulation, and antigen presentation were found to be altered significantly. These findings were replicated and compared with IFN-gamma-stimulated primary microglia using real-time quantitative RT-PCR validation. Importantly, a unique transcriptional phenotype with anti-inflammatory and IFN-gamma counter-regulatory properties partially related to alternatively activated macrophages was identified. Using functional cell assays, we found that CSPG-DS-stimulated microglia possess increased phagocytic capacity but lack direct cytotoxic effects such as secretion of NO. Furthermore, conditioned media from CSPG-DS-treated microglia did not diminish the viability or cause apoptosis of cultured photoreceptor cells and partially rescued these cells from IFN-gamma-induced apoptosis. Taken together, our data provide a unique transcript dataset and important in vitro findings about the functional properties of CSPG-DS-activated microglia. These might be starting points to explore the in vivo role of CSPG-DS as a bioactive microglia regulator and its potential, therapeutic application in immune-related, neurodegenerative disorders.
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Biomarcadores/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/farmacología , Microglía/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Perfilación de la Expresión Génica , Interferón gamma/farmacología , Ratones , Microglía/metabolismo , Óxido Nítrico/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fagocitosis/fisiología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
The RBP2-H1/JARID1B nuclear protein belongs to the ARID family of DNA-binding proteins and is a potential tumor suppressor that is lost during melanoma development. As we have recently shown, one physiological function of RBP2-H1/JARID1B is to exert cell cycle control via maintenance of active retinoblastoma protein. We now add new evidence that RBP2-H1/JARID1B can also directly regulate gene transcription in a reporter assay system, either alone or as part of a multimolecular complex together with the developmental transcription factors FOXG1b and PAX9. In melanoma cells, chromatin immunoprecipitation combined with promoter chip analysis (ChIP-on-chip) suggests a direct binding of re-expressed RBP2-H1/JARID1B to a multitude of human regulatory chromosomal elements (promoters, enhancers and introns). Among those, a set of 23 genes, including the melanoma relevant genes CDK6 and JAG-1 could be confirmed by cDNA microarray analyses to be differentially expressed after RBP2-H1/JARID1B re-expression. In contrast, in nonmelanoma HEK 293 cells, RBP2-H1/JARID1B overexpression only evokes a minor transcriptional response in cDNA microarray analyses. Because the transcriptional regulation in melanoma cells is accompanied by an inhibition of proliferation, an increase in caspase activity and a partial cell cycle arrest in G1/0, our data support an anti-tumorigenic role of RBP2-H1/JARID1B in melanocytic cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Melanoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Neoplasias Cutáneas/metabolismo , Transcripción Genética/fisiología , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Immunoblotting , Histona Demetilasas con Dominio de Jumonji , Melanoma/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Transcripción PAX9/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Represoras/genética , Neoplasias Cutáneas/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
There is increasing evidence that different phases of brain development depend on neuron-glia interactions including postnatal key events like synaptogenesis. To address how glial cells influence synapse development, we analyzed whether and how glia-derived factors affect gene expression in primary cultures of immunoisolated rat retinal ganglion cells (RGCs) by oligonucleotide microarrays. Our results show that the transcript pattern matched the developmental stage and characteristic properties of RGCs in vitro. Glia-conditioned medium (GCM) and cholesterol up- and downregulated a limited number of genes that influence the development of dendrites and synapses and regulate cholesterol and fatty acid metabolism. The oligonucleotide microarrays detected the transcriptional regulation of neuronal cholesterol homeostasis in response to GCM and cholesterol treatment. Surprisingly, our study revealed neuronal expression and glial regulation of matrix gla protein (Mgp). Together, our results suggest that glial cells promote different aspects of neuronal differentiation by regulating transcription of distinct classes of genes.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Animales , Antibacterianos , Western Blotting , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Diferenciación Celular/fisiología , Colesterol/metabolismo , Colesterol/farmacología , Medios de Cultivo Condicionados , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Ácidos Grasos/metabolismo , Filipina , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemo Oxigenasa (Desciclizante)/genética , Inmunohistoquímica , ARN/biosíntesis , ARN/genética , Ratas , Ratas Wistar , Células Ganglionares de la Retina/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinapsis/fisiología , Proteína Gla de la MatrizRESUMEN
AIM: To study susceptibility genes which may play a potential role in the pathogenesis and etiology of inflammatory bowel disease (IBD). METHODS: To identify potential susceptibility genes we performed global gene expression profiling in patients with IBD and control specimens. For determination of an intrinsic gene expression profile in ulcerative colitis (UC) and Crohn's disease (CD) compared to normal subjects, mucosal biopsies of non-inflamed regions of the colon and the terminal ileum were subjected to DNA microarray analysis. Real-time RT-PCR and immunohistochemistry were used for verification of selected regulated candidate genes and a genetic analysis was performed. RESULTS: We could show that aquaporin-8 (AQP8) mRNA and protein levels were significantly increased in the colon of UC patients compared to controls. Genetic analysis of the six exons and the promoter region of AQP8, however, revealed no mutations or polymorphisms in IBD patients. CONCLUSION: Our results suggest that upregulation of AQP8 in the colon of UC patients represents a secondary phenomenon which may, due to altered water exchange of the distal intestinal mucosa, disturb the physiologic colonic mucus barrier and thus lead to chronic inflammation and ulceration.
Asunto(s)
Acuaporinas/metabolismo , Colitis Ulcerosa/metabolismo , Colon/metabolismo , Íleon/metabolismo , Adulto , Acuaporinas/genética , Biopsia , Estudios de Casos y Controles , Colitis Ulcerosa/etiología , Colon/patología , Susceptibilidad a Enfermedades/metabolismo , Susceptibilidad a Enfermedades/patología , Femenino , Regulación de la Expresión Génica , Humanos , Íleon/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Conjugated Linoleic Acids (CLAs) are minor components of the diet with many reported biological activities. Our aim was to examine the function of the single trans-9,trans-11 (t9,t11), cis-9,trans-11 (c9,t11), and trans-10,cis-12 (t10,c12) isomers on gene expression in human macrophages. Therefore we incubated in vitro MCSF differentiated monocyte derived macrophages from three healthy donors and THP-1 macrophages with these CLA-isomers and analyzed whole genome transcripts with Affymetrix U133 Plus 2.0 DNA-microarrays and real-time RT-PCR. We found that t9,t11-CLA, but not c9,t11- and t10,c12-CLA activates target genes of SREBP, SREBP-1, and ABCG1. Gene reporter assays with deletion constructs of the ABCG1 regulatory region and cotransfections with SREBP-1a and SREBP-1c expression plasmids in RAW 264.7 macrophages showed that t9,t11-CLA activates ABCG1 via SREBP-1c. These results indicate that positional and geometrical isomers of CLAs have specific effects on gene expression of human macrophages and that t9,t11-CLA activates ABCG1 by a SREBP-1c-dependent mechanism.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ácidos Linoleicos Conjugados/administración & dosificación , Ácidos Linoleicos Conjugados/química , Macrófagos/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Activación Transcripcional/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Isomerismo , Macrófagos/efectos de los fármacos , Relación Estructura-Actividad , Activación Transcripcional/efectos de los fármacosRESUMEN
Loss of intestinal mucosa integrity is an important factor in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to characterize expression changes and allelic variants of genes related to intestinal epithelial barrier function in this disease. Therefore, ileal and colonic mucosal biopsies from nonaffected regions of patients with ulcerative colitis (UC) and Crohn's disease (CD), as well as non-IBD probands, were subjected to Affymetrix DNA-microarray analysis. Real-time reverse transcription polymerase chain reaction was used for verification in larger IBD sample numbers. Disturbed mRNA expression was identified for several mucin genes in both disease groups and tissues. A significant downregulation in the colon was obtained for MUC2 in CD and MUC12 in CD and UC. Expression analysis of all dysregulated mucins in a broad human tissue panel revealed dominant epithelial tissue-specific transcription. In silico analysis of the regulatory regions of these mucins indicated nuclear factor kappaB (NFkappaB) binding sites in each promoter. Furthermore, NFkappaB was overrepresented in mucin promoters and a component of a specific combination of transcription factors (composite module). In vivo stimulation experiments in the adenocarcinoma cell line LS174T showed inducible mucin expression by the cytokines tumor necrosis factor-alpha and transforming growth factor-beta, which could be blocked by NFkappaB signaling inhibitors. Allelic discrimination screening obtained statistically significant associations for the MUC2-V116M (P = 0.003) polymorphism with CD and for MUC4-A585S (P = 0.025), as well as MUC13-R502S (P = 0.0003) with UC. These data suggest that the disturbed expression of mucin genes and the connection to the NFkappaB pathway may influence the integrity of the intestine and therefore contribute to the pathophysiology of IBD.