RESUMO
Adaptive plasticity of Breast Cancer stem cells (BCSCs) is strongly correlated with cancer progression and resistance, leading to a poor prognosis. In this study, we report the expression profile of several pioneer transcription factors of the Oct3/4 network associated with tumor initiation and metastasis. In the triple negative breast cancer cell line (MDA-MB-231) stably transfected with human Oct3/4-GFP, differentially expressed genes (DEGs) were identified using qPCR and microarray, and the resistance to paclitaxel was assessed using an MTS assay. The tumor-seeding potential in immunocompromised (NOD-SCID) mice and DEGs in the tumors were also assessed along with the intra-tumor (CD44+/CD24-) expression using flow cytometry. Unlike 2-D cultures, the Oct3/4-GFP expression was homogenous and stable in 3-D mammospheres developed from BCSCs. A total of 25 DEGs including Gata6, FoxA2, Sall4, Zic2, H2afJ, Stc1 and Bmi1 were identified in Oct3/4 activated cells coupled with a significantly increased resistance to paclitaxel. In mice, the higher Oct3/4 expression in tumors correlated with enhanced tumorigenic potential and aggressive growth, with metastatic lesions showing a >5-fold upregulation of DEGs compared to orthotopic tumors and variability in different tissues with the highest modulation in the brain. Serially re-implanting tumors in mice as a model of recurrence and metastasis highlighted the sustained upregulation of Sall4, c-Myc, Mmp1, Mmp9 and Dkk1 genes in metastatic lesions with a 2-fold higher expression of stem cell markers (CD44+/CD24-). Thus, Oct3/4 transcriptome may drive the differentiation and maintenance of BCSCs, promoting their tumorigenic potential, metastasis and resistance to drugs such as paclitaxel with tissue-specific heterogeneity.
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Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Camundongos , Humanos , Animais , Feminino , Neoplasias da Mama/metabolismo , Regulação para Cima , Camundongos SCID , Camundongos Endogâmicos NOD , Neoplasias de Mama Triplo Negativas/patologia , Paclitaxel/farmacologia , Paclitaxel/metabolismo , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular TumoralRESUMO
Eosinophils, previously considered terminally differentiated effector cells, have multifaceted functions in tissues. We previously found that allergic mice with eosinophil-rich inflammation were protected from severe influenza and discovered specialized antiviral effector functions for eosinophils including promoting cellular immunity during influenza. In this study, we hypothesized that eosinophil responses during the early phase of influenza contribute to host protection. Using in vitro and in vivo models, we found that eosinophils were rapidly and dynamically regulated upon influenza A virus (IAV) exposure to gain migratory capabilities to traffic to lymphoid organs after pulmonary infection. Eosinophils were capable of neutralizing virus upon contact and combinations of eosinophil granule proteins reduced virus infectivity through hemagglutinin inactivation. Bi-directional crosstalk between IAV-exposed epithelial cells and eosinophils occurred after IAV infection and cross-regulation promoted barrier responses to improve antiviral defenses in airway epithelial cells. Direct interactions between eosinophils and airway epithelial cells after IAV infection prevented virus-induced cytopathology in airway epithelial cells in vitro, and eosinophil recipient IAV-infected mice also maintained normal airway epithelial cell morphology. Our data suggest that eosinophils are important in the early phase of IAV infection providing immediate protection to the epithelial barrier until adaptive immune responses are deployed during influenza.
Assuntos
Moléculas de Adesão Celular/metabolismo , Eosinófilos/metabolismo , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Vírus da Influenza A/genética , Animais , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
The 50 kDa N-terminal product of the cellular transcription factor E4F1 (p50E4F1) mediates E1A289R trans-activation of the adenovirus E4 gene, and suppresses E1A-mediated transformation by sensitizing cells to cell death. This report shows that while both E1A289R and E1A243R stimulate p50E4F1 DNA binding activity, E1A289R trans-activation, as measured using GAL-p50E4F1 fusion proteins, involves a p50E4F1 transcription regulatory (TR) region that must be promoter-bound and is dependent upon E1A CR3, CR1 and N-terminal domains. Trans-activation is promoter-specific, as GAL-p50E4F1 did not stimulate commonly used artificial promoters and was strongly repressive when competing against GAL-VP16. p50E4F1 and E1A289R stably associate in vivo using the p50E4F1 TR region and E1A CR3, although their association in vitro is indirect and paradoxically disrupted by MAP kinase phosphorylation of E1A289R, which stimulates E4 trans-activation in vivo. Multiple cellular proteins, including TBP, bind the p50E4F1 TR region in vitro. The mechanistic implications for p50E4F1 function are discussed.
Assuntos
Proteínas E1A de Adenovirus/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Ativação Transcricional , Ubiquitina-Proteína Ligases/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Sítios de Ligação , Células HEK293 , Células HeLa , Humanos , Fosforilação , Domínios Proteicos , Proteínas Repressoras/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases/genéticaRESUMO
Allergic asthma and influenza are common respiratory diseases with a high probability of co-occurrence. During the 2009 influenza pandemic, hospitalized patients with influenza experienced lower morbidity if asthma was an underlying condition. We have previously demonstrated that acute allergic asthma protects mice from severe influenza and have implicated eosinophils in the airways of mice with allergic asthma as participants in the antiviral response. However, very little is known about how eosinophils respond to direct exposure to influenza A virus (IAV) or the microenvironment in which the viral burden is high. We hypothesized that eosinophils would dynamically respond to the presence of IAV through phenotypic, transcriptomic, and physiologic changes. Using our mouse model of acute fungal asthma and influenza, we showed that eosinophils in lymphoid tissues were responsive to IAV infection in the lungs and altered surface expression of various markers necessary for cell activation in a niche-specific manner. Siglec-F expression was altered in a subset of eosinophils after virus exposure, and those expressing high Siglec-F were more active (IL-5Rαhi CD62Llo ). While eosinophils exposed to IAV decreased their overall transcriptional activity and mitochondrial oxygen consumption, transcription of genes encoding viral recognition proteins, Ddx58 (RIG-I), Tlr3, and Ifih1 (MDA5), were up-regulated. CD8+ T cells from IAV-infected mice expanded in response to IAV PB1 peptide-pulsed eosinophils, and CpG methylation in the Tbx21 promoter was reduced in these T cells. These data offer insight into how eosinophils respond to IAV and help elucidate alternative mechanisms by which they regulate antiviral immune responses during IAV infection.
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Eosinófilos/imunologia , Vírus da Influenza A/imunologia , Animais , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos Virais/imunologia , Asma/imunologia , Asma/patologia , Asma/virologia , Células da Medula Óssea/patologia , Linfócitos T CD8-Positivos/imunologia , Respiração Celular/genética , Galinhas , Desmetilação do DNA , Cães , Eosinófilos/metabolismo , Epigênese Genética , Feminino , Subunidade alfa de Receptor de Interleucina-5/metabolismo , Pulmão/patologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Fenótipo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Proteínas com Domínio T/metabolismo , Transcriptoma/genética , Regulação para CimaRESUMO
Urinary tract infections (UTI), associated with vesicoureteral reflux (VUR), can lead to chronic kidney disease. Genetic alterations in the innate immune defenses contribute to UTI risk. We investigated a novel gene, Dachsous Cadherin-Related 1 (DCHS1), in children with UTI. We determined absolute DNA copy number (CN) of DCHS1 in children with UTI. In this case-control study, we utilized multiple complementary methods to determine the genomic CN of DCHS1. Children with (n = 370) and without (n = 71) VUR from two well-phenotyped clinical trials of UTI were copy-typed and compared to 491 healthy controls with no known history of VUR or UTI. Less than 1% of controls had a single copy of DCHS1, while 31% of children with UTI and no VUR and 7% of children with UTI and VUR had a single copy of the DCHS1 gene. Using immunostaining, we localized expression postnatally to the bladder and renal epithelia. Mice were also challenged with two uropathogenic Escherichia coli strains, and Dchs1 mRNA was quantified. This study represents the first report of DCHS1 in association with pediatric UTI. We hypothesize that its role in innate immunity is critical to lower urinary tract defense. Further investigation is required to determine the role of DCHS1 in innate immunity.
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Caderinas/genética , Genótipo , Infecções Urinárias/genética , Animais , Proteínas Relacionadas a Caderinas , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RiscoRESUMO
Current healthcare is evolving to emphasize cost-effective care by leveraging results and outcomes of genomic and other advanced research efforts in clinical care and preventive health planning. Through a collaborative effort between the University of Tennessee Health Science Center (UTHSC) and Le Bonheur Children's Hospital (LBCH), the Biorepository and Integrative Genomics (BIG) Initiative was established to set up a pediatric-based DNA biorepository that can serve as a foundation for successful development of delivery platforms for predictive, preventive, and personalized medical services in Memphis, Tennessee, a historically disadvantaged community in the USA. In this paper, we describe the steps that were followed to establish the biorepository. We focused on domains that are essential for implementation of a biorepository for genomic research as an initial goal and identified patient consent, DNA extraction, storage and dissemination, and governance as essential components. Specific needs in each of these domains were addressed by respective solutions developed by multidisciplinary teams under the guidance of a governance model that involved experts from multiple hospital arenas and community members. The end result was the successful launch of a large-scale DNA biorepository, with patient consent greater than 75% in the first year. Our experience highlights the importance of performing pre-design research, needs assessment, and designing an ethically vetted plan that is cost-effective, easy to implement, and inclusive of the community that is served. We believe this biorepository model, with appropriate tailoring according to organizational needs and available resources, can be adopted and successfully applied by other small- to mid-sized healthcare organizations.
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Caregiving for a dementia patient is associated with increased risk of psychological and physical health problems. We investigated whether a mindfulness-based stress reduction (MBSR) training course for caregivers that closely models the MBSR curriculum originally established by the Center of Mindfulness at the University of Massachusetts may improve the psychological resilience of non-professional caregivers of Alzheimer's disease patients. Twenty adult non-professional caregivers of dementia patients participated in an 8-week MBSR training course. Caregiver stress, depression, burden, grief, and gene expression profiles of blood mononuclear cells were assessed at baseline and following MBSR. MBSR training significantly improved the psychological resilience of some of the caregivers. We identified predictive biomarkers whose expression is associated with the likelihood of caregivers to benefit from MBSR, and biomarkers whose expression is associated with MBSR psychological benefits. Our biomarker studies provide insight into the mechanisms of health benefits of MBSR and a basis for developing a personalized medicine approach for applying MBSR for promoting psychological and cognitive resilience in caregivers of dementia patients.
Assuntos
Biomarcadores/sangue , Cuidadores/educação , Cuidadores/psicologia , Atenção Plena , Estresse Psicológico/sangue , Estresse Psicológico/genética , Regulação da Expressão Gênica , Humanos , Atenção Plena/educação , Atenção Plena/normas , Estresse Psicológico/diagnóstico , TranscriptomaRESUMO
After demonstrating, with karyotyping, polymerase chain reaction (PCR) and fluorescence in-situ hybridization, the retention of certain human chromosomes and genes following the spontaneous fusion of human tumor and hamster cells in-vivo, it was postulated that cell fusion causes the horizontal transmission of malignancy and donor genes. Here, we analyzed gene expression profiles of 3 different hybrid tumors first generated in the hamster cheek pouch after human tumor grafting, and then propagated in hamsters and in cell cultures for years: two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749). Based on the criteria of MAS 5.0 detection P-values ≤0.065 and at least a 2-fold greater signal expression value than a hamster melanoma control, we identified 3,759 probe sets (ranging from 1,040 to 1,303 in each transplant) from formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors, which unambiguously mapped to 3,107 unique Entrez Gene IDs, representative of all human chromosomes; however, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing 33 unique Entrez Gene IDs, complied with the detection criteria in all hybrid tumor samples. Five of these 33 genes encode transcription factors that are known to regulate cell growth and differentiation; five encode cell adhesion- and transmigration-associated proteins that participate in oncogenesis and/or metastasis and invasion; and additional genes encode proteins involved in signaling pathways, regulation of apoptosis, DNA repair, and multidrug resistance. These findings were corroborated by PCR and reverse transcription PCR, showing the presence of human alphoid (α)-satellite DNA and the F11R transcripts in additional tumor transplant generations. We posit that in-vivo fusion discloses genes implicated in tumor progression, and gene families coding for the organoid phenotype. Thus, cancer cells can transduce adjacent stromal cells, with the resulting progeny having permanently transcribed genes with malignant and other gene functions of the donor DNA. Using heterospecific in-vivo cell fusion, genes encoding oncogenic and organogenic traits may be identified.
Assuntos
Fusão Celular , Linhagem Celular Tumoral , Células Estromais , Transcrição Gênica , Transdução Genética , Animais , Análise por Conglomerados , Cricetinae , Perfilação da Expressão Gênica , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
Disrupted synchronized oscillatory firing of pyramidal neuronal networks in the cerebral cortex in the gamma frequency band (i.e., 30-100 Hz) mediates many of the cognitive deficits and symptoms of schizophrenia. In fact, the density of dendritic spines and the average somal area of pyramidal neurons in layer 3 of the cerebral cortex, which mediate both long-range (associational) and local (intrinsic) corticocortical connections, are decreased in subjects with this illness. To explore the molecular pathophysiology of pyramidal neuronal dysfunction, we extracted ribonucleic acid (RNA) from laser-captured pyramidal neurons from layer 3 of Brodmann's area 42 of the superior temporal gyrus (STG) from postmortem brains from schizophrenia and normal control subjects. We then profiled the messenger RNA (mRNA) expression of these neurons, using microarray technology. We identified 1331 mRNAs that were differentially expressed in schizophrenia, including genes that belong to the transforming growth factor beta (TGF-ß) and the bone morphogenetic proteins (BMPs) signaling pathways. Disturbances of these signaling mechanisms may in part contribute to the altered expression of other genes found to be differentially expressed in this study, such as those that regulate extracellular matrix (ECM), apoptosis, and cytoskeletal and synaptic plasticity. In addition, we identified 10 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of their predicted gene targets revealed signaling pathways and gene networks that were found by microarray to be dysregulated, raising an interesting possibility that dysfunction of pyramidal neurons in schizophrenia may in part be mediated by a concerted dysregulation of gene network functions as a result of the altered expression of a relatively small number of miRNAs. Taken together, findings of this study provide a neurobiological framework within which specific hypotheses about the molecular mechanisms of pyramidal cell dysfunction in schizophrenia can be formulated.
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Células Piramidais/metabolismo , Esquizofrenia/genética , Esquizofrenia/patologia , Lobo Temporal/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Adulto JovemRESUMO
We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABAA) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABAA and GABAB subunits and overall reduced protein expression for GABAA receptor alpha 6 (GABRα6), GABAA receptor beta 2 (GABRß2), GABAA receptor delta (GABRδ), GABAA receptor epsilon (GABRε), GABAA receptor gamma 2 (GABRγ2), GABAA receptor theta (GABRθ), and GABAA receptor rho 2 (GABRρ2) in superior frontal cortex from subjects with autism. Our data demonstrate systematic changes in GABAA&B subunit expression in brains of subjects with autism, which may help explain the presence of cognitive abnormalities in subjects with autism.
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Transtorno Autístico/metabolismo , Regulação para Baixo , Lobo Frontal/metabolismo , Receptores de GABA-A/genética , Adulto , Animais , Transtorno Autístico/genética , Estudos de Casos e Controles , Cerebelo/metabolismo , Feminino , Expressão Gênica , Humanos , Masculino , Lobo Parietal/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de GABA-A/metabolismo , Adulto Jovem , Ácido gama-Aminobutírico/metabolismoRESUMO
Dysregulation of pyramidal cell network function by the soma- and axon-targeting inhibitory neurons that contain the calcium-binding protein parvalbumin (PV) represents a core pathophysiological feature of schizophrenia. In order to gain insight into the molecular basis of their functional impairment, we used laser capture microdissection (LCM) to isolate PV-immunolabeled neurons from layer 3 of Brodmann's area 42 of the superior temporal gyrus (STG) from postmortem schizophrenia and normal control brains. We then extracted ribonucleic acid (RNA) from these neurons and determined their messenger RNA (mRNA) expression profile using the Affymetrix platform of microarray technology. Seven hundred thirty-nine mRNA transcripts were found to be differentially expressed in PV neurons in subjects with schizophrenia, including genes associated with WNT (wingless-type), NOTCH, and PGE2 (prostaglandin E2) signaling, in addition to genes that regulate cell cycle and apoptosis. Of these 739 genes, only 89 (12%) were also differentially expressed in pyramidal neurons, as described in the accompanying paper, suggesting that the molecular pathophysiology of schizophrenia appears to be predominantly neuronal type specific. In addition, we identified 15 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of the predicted targets of these miRNAs included the signaling pathways found by microarray to be dysregulated in schizophrenia. Taken together, findings of this study provide a neurobiological framework within which hypotheses of the molecular mechanisms that underlie the dysfunction of PV neurons in schizophrenia can be generated and experimentally explored and, as such, may ultimately inform the conceptualization of rational targeted molecular intervention for this debilitating disorder.
Assuntos
Neurônios/metabolismo , Parvalbuminas/genética , Parvalbuminas/metabolismo , Esquizofrenia , Lobo Temporal/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Calbindinas/metabolismo , Dinoprostona/genética , Dinoprostona/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Receptor Notch1/genética , Receptor Notch1/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Transdução de Sinais/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Adulto JovemRESUMO
Dopamine (DA) neurons in sporadic Parkinson's disease (PD) display dysregulated gene expression networks and signaling pathways that are implicated in PD pathogenesis. Micro (mi)RNAs are regulators of gene expression, which could be involved in neurodegenerative diseases. We determined the miRNA profiles in laser microdissected DA neurons from postmortem sporadic PD patients' brains and age-matched controls. DA neurons had a distinctive miRNA signature and a set of miRNAs was dysregulated in PD. Bioinformatics analysis provided evidence for correlations of miRNAs with signaling pathways relevant to PD, including an association of miR-126 with insulin/IGF-1/PI3K signaling. In DA neuronal cell systems, enhanced expression of miR-126 impaired IGF-1 signaling and increased vulnerability to the neurotoxin 6-OHDA by downregulating factors in IGF-1/PI3K signaling, including its targets p85ß, IRS-1, and SPRED1. Blocking of miR-126 function increased IGF-1 trophism and neuroprotection to 6-OHDA. Our data imply that elevated levels of miR-126 may play a functional role in DA neurons and in PD pathogenesis by downregulating IGF-1/PI3K/AKT signaling and that its inhibition could be a mechanism of neuroprotection.
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Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica/genética , Fator de Crescimento Insulin-Like I/metabolismo , MicroRNAs/fisiologia , Doença de Parkinson/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Encéfalo/metabolismo , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Humanos , Oxidopamina/toxicidade , Doença de Parkinson/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
BACKGROUND: The present study was designed to validate the ability of our recently identified set of small noncoding RNA candidate mild traumatic brain injury (mTBI) biomarkers to diagnose mTBI in the presence or absence of post-traumatic stress disorder (PTSD) comorbidity. Using qPCR, we explored the regulation of the candidate biomarkers in peripheral blood mononuclear cells (PBMC) from 58 veterans. RESULTS: We confirmed that 4 small nucleolar RNAs (snoRNAs), ACA48, U35, U55, and U83A, are significantly down-regulated in PBMC from veterans with mTBI and PTSD compared to non-TBI, control subjects with PTSD only. We found that the snoRNA biomarkers are able to dissect subjects with comorbid mTBI and PTSD from PTSD subjects without mTBI with 100% sensitivity, 81% accuracy, and 72% specificity. No significant differential expression of snoRNA biomarkers was found in mTBI subjects without comorbid PTSD. However, we found significantly lower U55 contents in subjects with PTSD. We explored the regulation of ACA48 in rodent models of PTSD or blast-induced mTBI to gather proof-of-concept evidence that would connect the regulation of the biomarkers and the development of mTBI or PTSD. We found no change in the regulation of ACA48 in the mTBI rat model. We did, however, find significant down-regulation of ACA48 in the PTSD mouse model 24 hours following psychological trauma exposure. This may reflect a short-term response to trauma exposure, since we found no change in the regulation of ACA48 in veteran PTSD subjects 3.6 years post-deployment. CONCLUSIONS: Additional application of the 4 snoRNA biomarker to current diagnostic criteria may provide an objective biomarker pattern to help identify veterans with comorbid mTBI and PTSD. Our observations suggest that biological interactions between TBI and PTSD may contribute to the clinical features of veterans with comorbid mTBI and PTSD. Future investigations on mTBI mechanisms or TBI biomarkers should consider their interactions with PTSD.
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Repeated exposure to cocaine induces neuroadaptations which contribute to the rewarding properties of cocaine. Using cocaine-induced conditioned place preference (CPP) as an animal model of reward, earlier studies have shown that sigma (σ) receptor ligands can attenuate the acquisition, expression and reactivation of CPP. However, the underlying molecular mechanisms that are associated with these changes are not yet understood. In the present study, CM156, a novel antagonist with high selectivity and affinity for σ receptors was used to attenuate the expression of cocaine-induced CPP in mice. Immediately following the behavioral evaluations, mouse brain tissues were collected and alterations in gene expression in half brain samples were profiled by cDNA microarray analysis. Microarray data was analyzed by three distinct normalization methods and four genes were consistently found to be upregulated by cocaine when compared to saline controls. Each of these gene changes were found by more than one normalization method to be reversed by at least one dose of CM156. Quantitative real time PCR confirmed that a single administration of CM156 was able to reverse the cocaine-induced increases in three of these four genes: metastasis associated lung adenocarcinoma transcript 1 (malat1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (ywhaz), and transthyretin (ttr). These genes are involved in processes related to neuroplasticity and RNA editing. The data presented herein provides evidence that pharmacological intervention with a putative σ receptor antagonist reverses alterations in gene expression that are associated with cocaine-induced reward.
Assuntos
Proteínas 14-3-3/genética , Química Encefálica/efeitos dos fármacos , Química Encefálica/genética , Cocaína/antagonistas & inibidores , Condicionamento Operante/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Piperazinas/farmacologia , Receptores sigma/antagonistas & inibidores , Compostos de Enxofre/farmacologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Cocaína/farmacologia , DNA Complementar/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Atividade Motora/efeitos dos fármacos , Pré-Albumina/genética , RNA/biossíntese , RNA/isolamento & purificação , RNA Longo não Codificante , RNA não Traduzido/genética , Reação em Cadeia da Polimerase em Tempo Real , Esquema de Reforço , Recompensa , Convulsões/induzido quimicamenteRESUMO
Researchers have long noted an excess of patients with schizophrenia were born during the months of January and March. This winter birth effect has been hypothesized to result either from various causes such as vitamin D deficiency (McGrath, 1999; McGrath et al., 2010), or from maternal infection during pregnancy. Infection with a number of viruses during pregnancy including influenza, and rubella are known to increase the risk of schizophrenia in the offspring (Brown, 2006). Animal models using influenza virus or Poly I:C, a viral mimic, have been able to replicate many of the brain morphological, genetic, and behavioral deficits of schizophrenia (Meyer et al., 2006, 2008a, 2009; Bitanihirwe et al., 2010; Meyer and Feldon, 2010; Short et al., 2010). Using a murine model of prenatal viral infection, our laboratory has shown that viral infection on embryonic days 9, 16, and 18 leads to abnormal expression of brain genes and brain structural abnormalities in the exposed offspring (Fatemi et al., 2005, 2008a,b, 2009a,b). The purpose of the current study was to examine gene expression and morphological changes in the placenta, hippocampus, and prefrontal cortex as a result of viral infection on embryonic day 7 of pregnancy. Pregnant mice were either infected with influenza virus [A/WSN/33 strain (H1N1)] or sham-infected with vehicle solution. At E16, placentas were harvested and prepared for either microarray analysis or for light microscopy. We observed significant, upregulation of 77 genes and significant downregulation of 93 genes in placentas. In brains of exposed offspring following E7 infection, there were changes in gene expression in prefrontal cortex (6 upregulated and 24 downregulated at P0; 5 upregulated and 14 downregulated at P56) and hippocampus (4 upregulated and 6 downregulated at P0; 6 upregulated and 13 downregulated at P56). QRT-PCR verified the direction and magnitude of change for a number of genes associated with hypoxia, inflammation, schizophrenia, and autism. Placentas from infected mice showed a number of morphological abnormalities including presence of thrombi and increased presence of immune cells. Additionally, we searched for presence of H1N1 viral-specific genes for M1/M2, NA, and NS1 in placentas of infected mice and brains of exposed offspring and found none. Our results demonstrate that prenatal viral infection disrupts structure and gene expression of the placenta, hippocampus, and prefrontal cortex potentially explaining deleterious effects in the exposed offspring without evidence for presence of viral RNAs in the target tissues.
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Encéfalo/virologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Viral da Expressão Gênica , Vírus da Influenza A Subtipo H1N1/genética , Placenta/virologia , Esquizofrenia/genética , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/patologia , Placenta/metabolismo , Placenta/patologia , Gravidez , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Esquizofrenia/virologiaRESUMO
We investigated the genome-wide consequences of pan-histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and m-carboxycinnamic acid bis-hydroxamide (CBHA) in the hearts of BALB/c mice eliciting hypertrophy in response to interleukin-18 (IL-18). Both TSA and CBHA profoundly altered cardiac chromatin structure that occurred concomitantly with normalization of IL-18-induced gene expression and amelioration of cardiac hypertrophy. The hearts of mice exposed to IL-18+/-TSA or CBHA elicited distinct gene expression profiles. Of 184 genes that were differentially regulated by IL-18 and TSA, 33 were regulated in an opposite manner. The hearts of mice treated with IL-18 and/or CBHA elicited 147 differentially expressed genes (DEGs), a third of which were oppositely regulated by IL-18 and CBHA. Ingenuity Pathways and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs showed that IL-18 impinged on TNF-α- and IFNγ-specific gene networks relegated to controlling immunity and inflammation, cardiac metabolism and energetics, and cell proliferation and apoptosis. These TNF-α- and IFNγ-specific gene networks, extensively connected with PI3K, MAPK, and NF-κB signaling pathways, were oppositely regulated by IL-18 and pan-HDACIs. Evidently, both TSA and CBHA caused a two- to fourfold induction of phosphatase and tensin homolog expression to counteract IL-18-induced proinflammatory signaling and cardiac hypertrophy.
Assuntos
Cardiomegalia/tratamento farmacológico , Cardiomegalia/patologia , Inibidores de Histona Desacetilases/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-18/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Cardiomegalia/complicações , Cardiomegalia/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Inflamação/complicações , Inflamação/tratamento farmacológico , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/metabolismo , Miocárdio/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Reprodutibilidade dos Testes , Transdução de Sinais/genéticaRESUMO
We have shown altered expression of gamma-aminobutyric acid A (GABA(A)) and gamma-aminobutyric acid B (GABA(B)) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3 GABA(A) subunits previously associated with autism (GABRalpha4; GABRalpha5; GABRbeta1). Three GABA receptor subunits demonstrated mRNA and protein level concordance in superior frontal cortex (GABRalpha4, GABRalpha5, GABRbeta1) and one demonstrated concordance in cerebellum (GABBetaR1). These results provide further evidence of impairment of GABAergic signaling in autism.
Assuntos
Transtorno Autístico/metabolismo , Química Encefálica , Receptores de GABA-A/análise , Receptores de GABA-B/análise , Adulto , Western Blotting , Cerebelo/química , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Bone Morphogenetic Proteins (BMPs) are members of the TGF-beta superfamily of growth factors. Several BMPs exhibit osteoinductive bioactivities, and are critical for bone formation in both developing and mature skeletal systems. BMP-7 (OP-1) is currently used clinically in revision of posterolateral spine fusions and long bone non-unions. The current study characterizes BMP-7 induced gene expression during early osteoblastic differentiation of human mesenchymal stem cells (hMSC). Primary hMSC were treated with BMP-7 for 24 or 120 h and gene expression across the entire human genome was evaluated using Affymetrix HG-U133 Plus 2.0 Arrays. 955 probe sets representing 655 genes and 95 ESTs were identified as differentially expressed and were organized into three major expression profiles (Profiles A, B and C) by hierarchical clustering. Genes from each profile were classified according to biochemical pathway analyses. Profile A, representing genes upregulated by BMP-7, revealed strong enrichment for established osteogenic marker genes, as well as several genes with undefined roles in osteoblast function, including MFI2, HAS3, ADAMTS9, HEY1, DIO2 and FGFR3. A functional screen using siRNA suggested roles for MFI2, HEY1 and DIO2 in osteoblastic differentiation of hMSC. Profile B contained genes transiently downregulated by BMP-7, including numerous genes associated with cell cycle regulation. Follow-up studies confirmed that BMP-7 attenuates cell cycle progression and cell proliferation during early osteoblastic differentiation. Profile C, comprised of genes continuously downregulated by BMP-7, exhibited strong enrichment for genes associated with chemokine/cytokine activity. Inhibitory effects of BMP-7 on cytokine secretion were verified by analysis of enriched culture media. Potent downregulation of CHI3L1, a potential biomarker for numerous joint diseases, was also observed in Profile C. A focused evaluation of BMP, GDF and BMP inhibitor expression elucidated feedback loops modulating BMP-7 bioactivity. BMP-7 was found to induce BMP-2 and downregulate GDF5 expression. Transient knockdown of BMP-2 using siRNA demonstrated that osteoinductive properties associated with BMP-7 are independent of endogenous BMP-2 expression. Noggin was identified as the predominant inhibitor induced by BMP-7 treatment. Overall, this study provides new insight into key bioactivities characterizing early BMP-7 mediated osteoblastic differentiation.
Assuntos
Proteína Morfogenética Óssea 7/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Adipocinas , Adolescente , Adulto , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Células Cultivadas , Proteína 1 Semelhante à Quitinase-3 , Análise por Conglomerados , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/metabolismo , Humanos , Lectinas , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Osteoblastos/metabolismo , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The objective of this study was to determine the molecular bases of disordered hepatic function and disease susceptibility in obesity. We compared global gene expression in liver biopsies from morbidly obese (MO) women undergoing gastric bypass (GBP) surgery with that of women undergoing ventral hernia repair who had experienced massive weight loss (MWL) following prior GBP. Metabolic and hormonal profiles were examined in MO vs. MWL groups. Additionally, we analyzed individual profiles of hepatic gene expression in liver biopsy specimens obtained from MO and MWL subjects. All patients underwent preoperative metabolic profiling. RNAs were extracted from wedge biopsies of livers from MO and MWL subjects, and analysis of mRNA expression was carried out using Affymetrix HG-U133A microarray gene chips. Genes exhibiting greater than twofold differential expression between MO and MWL subjects were organized according to gene ontology and hierarchical clustering, and expression of key genes exhibiting differential regulation was quantified by real-time-polymerase chain reaction (RT-PCR). We discovered 154 genes to be differentially expressed in livers of MWL and MO subjects. A total of 28 candidate disease susceptibility genes were identified that encoded proteins regulating lipid and energy homeostasis (PLIN, ENO3, ELOVL2, APOF, LEPR, IGFBP1, DDIT4), signal transduction (MAP2K6, SOCS-2), postinflammatory tissue repair (HLA-DQB1, SPP1, P4HA1, LUM), bile acid transport (SULT2A, ABCB11), and metabolism of xenobiotics (GSTT2, CYP1A1). Using gene expression profiling, we have identified novel candidate disease susceptibility genes whose expression is altered in livers of MO subjects. The significance of altered expression of these genes to obesity-related disease is discussed.
Assuntos
Regulação da Expressão Gênica , Predisposição Genética para Doença , Fígado/metabolismo , Obesidade Mórbida/genética , Adulto , Biópsia , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação , Metabolismo dos Lipídeos , Fígado/patologia , Obesidade Mórbida/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The leucine-rich acidic nuclear protein (LANP) belongs to the INHAT family of corepressors that inhibits histone acetyltransferases. The mechanism by which LANP restricts its repression to specific genes is unknown. Here, we report that LANP forms a complex with transcriptional repressor E4F and modulates its activity. As LANP interacts with ataxin 1--a protein mutated in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1)--we tested whether ataxin 1 can alter the E4F-LANP interaction. We show that ataxin 1 relieves the transcriptional repression induced by the LANP-E4F complex by competing with E4F for LANP. These results provide the first functional link, to our knowledge, between LANP and ataxin 1, and indicate a potential mechanism for the transcriptional aberrations observed in SCA1.