RESUMO
BACKGROUND: Coronary artery calcification (CAC) is a noninvasive measure of coronary atherosclerosis, the proximal pathophysiology underlying most cases of myocardial infarction (MI). We sought to identify expression signatures of early MI and subclinical atherosclerosis in the Framingham Heart Study (FHS). In this study, we conducted paired-end RNA sequencing on whole blood collected from 198 FHS participants (55 with a history of early MI, 72 with high CAC without prior MI, and 71 controls free of elevated CAC levels or history of MI). We applied DESeq2 to identify coding-genes and long intergenic noncoding RNAs (lincRNAs) differentially expressed in MI and high CAC, respectively, compared with the control. RESULTS: On average, 150 million paired-end reads were obtained for each sample. At the false discovery rate (FDR) < 0.1, we found 68 coding genes and 2 lincRNAs that were differentially expressed in early MI versus controls. Among them, 60 coding genes were detectable and thus tested in an independent RNA-Seq data of 807 individuals from the Rotterdam Study, and 8 genes were supported by p value and direction of the effect. Immune response, lipid metabolic process, and interferon regulatory factor were enriched in these 68 genes. By contrast, only 3 coding genes and 1 lincRNA were differentially expressed in high CAC versus controls. APOD, encoding a component of high-density lipoprotein, was significantly downregulated in both early MI (FDR = 0.007) and high CAC (FDR = 0.01) compared with controls. CONCLUSIONS: We identified transcriptomic signatures of early MI that include differentially expressed protein-coding genes and lincRNAs, suggesting important roles for protein-coding genes and lincRNAs in the pathogenesis of MI.
Assuntos
Doença da Artéria Coronariana , Humanos , Infarto do Miocárdio , RNA Longo não Codificante/genética , Análise de Sequência de RNA , Transcriptoma , Sequenciamento do ExomaRESUMO
Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNß stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of â¼2,000 IFNß-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.
Assuntos
Células da Medula Óssea/imunologia , Divisão Celular/imunologia , Epigênese Genética/imunologia , Imunidade Inata , Interferon beta/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Transcrição Gênica/imunologia , Animais , Células da Medula Óssea/citologia , Divisão Celular/genética , Cromatina/genética , Cromatina/imunologia , Histonas/genética , Histonas/imunologia , Interferon beta/genética , Macrófagos/citologia , Camundongos , Camundongos Mutantes , RNA Polimerase II/genética , RNA Polimerase II/imunologia , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Bones at different anatomical locations vary dramatically in size. For example, human femurs are 20-fold longer than the phalanges in the fingers and toes. The mechanisms responsible for these size differences are poorly understood. Bone elongation occurs at the growth plates and advances rapidly in early life but then progressively slows due to a developmental program termed "growth plate senescence." This developmental program includes declines in cell proliferation and hypertrophy, depletion of cells in all growth plate zones, and extensive underlying changes in the expression of growth-regulating genes. Here, we show evidence that these functional, structural, and molecular senescent changes occur earlier in the growth plates of smaller bones (metacarpals, phalanges) than in the growth plates of larger bones (femurs, tibias) and that this differential aging contributes to the disparities in bone length. We also show evidence that the molecular mechanisms that underlie the differential aging between different bones involve modulation of critical paracrine regulatory pathways, including insulin-like growth factor (Igf), bone morphogenetic protein (Bmp), and Wingless and Int-1 (Wnt) signaling. Taken together, the findings reveal that the striking disparities in the lengths of different bones, which characterize normal mammalian skeletal proportions, is achieved in part by modulating the progression of growth plate senescence.
Assuntos
Envelhecimento/fisiologia , Osso e Ossos/anatomia & histologia , Cartilagem/crescimento & desenvolvimento , Lâmina de Crescimento/crescimento & desenvolvimento , Animais , Desenvolvimento Ósseo , Proliferação de Células , Condrócitos/patologia , Extremidades/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Hipertrofia , Camundongos Endogâmicos C57BL , Comunicação Parácrina , Ratos Sprague-Dawley , Tíbia/crescimento & desenvolvimentoRESUMO
Genome-wide association studies (GWAS) have highlighted a large number of genetic variants with potential disease association, but functional analysis remains a challenge. Here we describe an approach to functionally validate identified variants through differentiation of induced pluripotent stem cells (iPSCs) to study cellular pathophysiology. We collected peripheral blood cells from Framingham Heart Study participants and reprogrammed them to iPSCs. We then differentiated 68 iPSC lines into hepatocytes and adipocytes to investigate the effect of the 1p13 rs12740374 variant on cardiometabolic disease phenotypes via transcriptomics and metabolomic signatures. We observed a clear association between rs12740374 and lipid accumulation and gene expression in differentiated hepatocytes, in particular, expression of SORT1, CELSR2, and PSRC1, consistent with previous analyses of this variant using other approaches. Initial investigation of additional SNPs also highlighted correlations with gene expression. These findings suggest that iPSC-based population studies hold promise as tools for the functional validation of GWAS variants.
Assuntos
Diferenciação Celular/genética , Estudo de Associação Genômica Ampla , Células-Tronco Pluripotentes Induzidas/citologia , Doenças Metabólicas/genética , Adipócitos Brancos/citologia , Adipócitos Brancos/metabolismo , Reprogramação Celular/genética , Cromossomos Humanos Par 1/genética , Estudos de Coortes , Regulação para Baixo/genética , Genótipo , Hepatócitos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Metabolismo dos Lipídeos/genética , Metabolômica , Modelos Genéticos , Fenótipo , Locos de Características Quantitativas/genética , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Doadores de Tecidos , Transcriptoma/genéticaRESUMO
In this mini-review, the role of macrophage phenotypes in atherogenesis is considered. Recent studies on distribution of M1 and M2 macrophages in different types of atherosclerotic lesions indicate that macrophages exhibit a high degree of plasticity of phenotype in response to various conditions in microenvironment. The effect of the accumulation of cholesterol, a key event in atherogenesis, on the macrophage phenotype is also discussed. The article presents the results of transcriptome analysis of cholesterol-loaded macrophages revealing genes involved in immune response whose expression rate has changed the most. It turned out that the interaction of macrophages with modified LDL leads to higher expression levels of pro-inflammatory marker TNF-α and antiinflammatory marker CCL18. Phenotypic profile of macrophage activation could be a good target for testing of novel anti-atherogenic immunocorrectors. A number of anti-atherogenic drugs were tested as potential immunocorrectors using primary macrophage-based model.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticolesterolemiantes/farmacologia , Aterosclerose/tratamento farmacológico , Aterosclerose/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Animais , HumanosRESUMO
BACKGROUND: Studies of thyroid stem/progenitor cells have been hampered due to the small organ size and lack of tissue, which limits the yield of these cells. A continuous source that allows the study and characterization of thyroid stem/progenitor cells is desired to push the field forward. METHOD: A cell line was established from Hoechst-resistant side population cells derived from mouse thyroid that were previously shown to contain stem/progenitor-like cells. Characterization of these cells were carried out by using in vitro two- and three-dimensional cultures and in vivo reconstitution of mice after orthotopic or intravenous injection, in conjunction with quantitative reverse transcription polymerase chain reaction, Western blotting, immunohisto(cyto)chemistry/immunofluorescence, and RNA seq analysis. RESULTS: These cells were named SPTL (side population cell-derived thyroid cell line). Under low serum culturing conditions, SPTL cells expressed the thyroid differentiation marker NKX2-1, a transcription factor critical for thyroid differentiation and function, while no expression of other thyroid differentiation marker genes were observed. SPTL cells formed follicle-like structures in Matrigel® cultures, which did not express thyroid differentiation marker genes. In mouse models of orthotopic and intravenous injection, the latter following partial thyroidectomy, a few SPTL cells were found in part of the follicles, most of which expressed NKX2-1. SPTL cells highly express genes involved in epithelial-mesenchymal transition, as demonstrated by RNA seq analysis, and exhibit a gene-expression pattern similar to anaplastic thyroid carcinoma. CONCLUSION: These results demonstrate that SPTL cells have the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid.
Assuntos
Diferenciação Celular , Transição Epitelial-Mesenquimal , Células da Side Population/citologia , Células-Tronco/citologia , Glândula Tireoide/citologia , Animais , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular , Imunofluorescência , Expressão Gênica , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Células da Side Population/metabolismo , Transplante de Células-Tronco , Células-Tronco/metabolismo , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , TireoidectomiaRESUMO
SCOPE: Fish oil-derived long-chain monounsaturated fatty acids (LCMUFA) containing chain lengths longer than 18 were previously shown to improve cardiovascular disease risk factors in mice. However, it is not known if LCMUFA also exerts anti-atherogenic effects. The main objective of the present study was to investigate the effect of LCMUFA on the development of atherosclerosis in mouse models. METHODS AND RESULTS: LDLR-KO mice were fed Western diet supplemented with 2% (w/w) of either LCMUFA concentrate, olive oil, or not (control) for 12 wk. LCMUFA, but not olive oil, significantly suppressed the development of atherosclerotic lesions and several plasma inflammatory cytokine levels, although there were no major differences in plasma lipids between the three groups. At higher doses 5% (w/w) LCMUFA supplementation was observed to reduce pro-atherogenic plasma lipoproteins and to also reduce atherosclerosis in ApoE-KO mice fed a Western diet. RNA sequencing and subsequent qPCR analyses revealed that LCMUFA upregulated PPAR signaling pathways in liver. In cell culture studies, apoB-depleted plasma from LDLR-K mice fed LCMUFA showed greater cholesterol efflux from macrophage-like THP-1 cells and ABCA1-overexpressing BHK cells. CONCLUSION: Our research showed for the first time that LCMUFA consumption protects against diet-induced atherosclerosis, possibly by upregulating the PPAR signaling pathway.
Assuntos
Aterosclerose/prevenção & controle , Ácidos Graxos Monoinsaturados/farmacologia , Óleos de Peixe/farmacologia , Animais , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Linhagem Celular , Colesterol/metabolismo , Citocinas/sangue , Modelos Animais de Doenças , Ácidos Graxos/análise , Ácidos Graxos Monoinsaturados/química , Óleos de Peixe/química , Humanos , Lipídeos/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Knockout , Receptores de LDL/genéticaRESUMO
T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Fator de Transcrição AP-1/metabolismo , Vaccinia virus/imunologia , Vacínia/imunologia , Imunidade Adaptativa , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD8-Positivos/virologia , Diferenciação Celular/genética , Células Cultivadas , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Memória Imunológica/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Oncogênica p65(gag-jun) , Transdução de Sinais/genética , Fator de Transcrição AP-1/genéticaRESUMO
BACKGROUND & AIMS: A genome-wide association study (GWAS) of 280 cases identified the hepatic cholesterol transporter ABCG8 as a locus associated with risk for gallstone disease, but findings have not been reported from any other GWAS of this phenotype. We performed a large-scale, meta-analysis of GWASs of individuals of European ancestry with available prior genotype data, to identify additional genetic risk factors for gallstone disease. METHODS: We obtained per-allele odds ratio (OR) and standard error estimates using age- and sex-adjusted logistic regression models within each of the 10 discovery studies (8720 cases and 55,152 controls). We performed an inverse variance weighted, fixed-effects meta-analysis of study-specific estimates to identify single-nucleotide polymorphisms that were associated independently with gallstone disease. Associations were replicated in 6489 cases and 62,797 controls. RESULTS: We observed independent associations for 2 single-nucleotide polymorphisms at the ABCG8 locus: rs11887534 (OR, 1.69; 95% confidence interval [CI], 1.54-1.86; P = 2.44 × 10(-60)) and rs4245791 (OR, 1.27; P = 1.90 × 10(-34)). We also identified and/or replicated associations for rs9843304 in TM4SF4 (OR, 1.12; 95% CI, 1.08-1.16; P = 6.09 × 10(-11)), rs2547231 in SULT2A1 (encodes a sulfoconjugation enzyme that acts on hydroxysteroids and cholesterol-derived sterol bile acids) (OR, 1.17; 95% CI, 1.12-1.21; P = 2.24 × 10(-10)), rs1260326 in glucokinase regulatory protein (OR, 1.12; 95% CI, 1.07-1.17; P = 2.55 × 10(-10)), and rs6471717 near CYP7A1 (encodes an enzyme that catalyzes conversion of cholesterol to primary bile acids) (OR, 1.11; 95% CI, 1.08-1.15; P = 8.84 × 10(-9)). Among individuals of African American and Hispanic American ancestry, rs11887534 and rs4245791 were associated positively with gallstone disease risk, whereas the association for the rs1260326 variant was inverse. CONCLUSIONS: In this large-scale GWAS of gallstone disease, we identified 4 loci in genes that have putative functions in cholesterol metabolism and transport, and sulfonylation of bile acids or hydroxysteroids.
Assuntos
Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Cálculos Biliares/genética , Loci Gênicos/genética , Predisposição Genética para Doença , Adulto , Negro ou Afro-Americano/genética , Idoso , Estudos de Casos e Controles , Colesterol/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Hispânico ou Latino/genética , Humanos , Metabolismo dos Lipídeos/genética , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Fenótipo , Polimorfismo de Nucleotídeo Único , População Branca/genéticaRESUMO
Polyfunctionality and cytotoxic activity dictate CD8(+) T-cell efficacy in the eradication of infected and malignant cells. The induction of these effector functions depends on the specific interaction between the T-cell receptor (TCR) and its cognate peptide-MHC class I complex, in addition to signals provided by co-stimulatory or co-inhibitory receptors, which can further regulate these functions. Among these receptors, the role of 2B4 is contested, as it has been described as either co-stimulatory or co-inhibitory in modulating T-cell functions. We therefore combined functional, transcriptional and epigenetic approaches to further characterize the impact of disrupting the interaction of 2B4 with its ligand CD48, on the activity of human effector CD8(+) T-cell clones. In this setting, we show that the 2B4-CD48 axis is involved in the fine-tuning of CD8(+) T-cell effector function upon antigenic stimulation. Blocking this interaction resulted in reduced CD8(+) T-cell clone-mediated cytolytic activity, together with a subtle drop in the expression of genes involved in effector function regulation. Our results also imply a variable contribution of the 2B4-CD48 interaction to the modulation of CD8(+) T-cell functional properties, potentially linked to intrinsic levels of T-bet expression and TCR avidity. The present study thus provides further insights into the role of the 2B4-CD48 interaction in the fine regulation of CD8(+) T-cell effector function upon antigenic stimulation.
Assuntos
Antígeno CD48/metabolismo , Linfócitos T CD8-Positivos/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Afinidade de Anticorpos/imunologia , Citotoxicidade Imunológica/genética , Epigênese Genética , Humanos , Imunomodulação , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteínas com Domínio T/metabolismo , Transcrição GênicaRESUMO
Transcripts in platelets are largely produced in precursor megakaryocytes but remain physiologically active as platelets translate RNAs and regulate protein/RNA levels. Recent studies using transcriptome sequencing (RNA-seq) characterized the platelet transcriptome in limited number of non-diseased individuals. Here, we expand upon these RNA-seq studies by completing RNA-seq in platelets from 32 patients with acute myocardial infarction (MI). Our goals were to characterize the platelet transcriptome using a population of patients with acute MI and relate gene expression to platelet aggregation measures and ST-segment elevation MI (STEMI) (n = 16) vs. non-STEMI (NSTEMI) (n = 16) subtypes. Similar to other studies, we detected 9565 expressed transcripts, including several known platelet-enriched markers (e.g. PPBP, OST4). Our RNA-seq data strongly correlated with independently ascertained platelet expression data and showed enrichment for platelet-related pathways (e.g. wound response, hemostasis, and platelet activation), as well as actin-related and post-transcriptional processes. Several transcripts displayed suggestively higher (FBXL4, ECHDC3, KCNE1, TAOK2, AURKB, ERG, and FKBP5) and lower (MIAT, PVRL3, and PZP) expression in STEMI platelets compared to NSTEMI. We also identified transcripts correlated with platelet aggregation to TRAP (ATP6V1G2, SLC2A3), collagen (CEACAM1, ITGA2), and ADP (PDGFB, PDGFC, ST3GAL6). Our study adds to current platelet gene expression resources by providing transcriptome-wide analyses in platelets isolated from patients with acute MI. In concert with prior studies, we identify various genes for further study in regards to platelet function and acute MI. Future platelet RNA-seq studies examining more diverse sets of healthy and diseased samples will add to our understanding of platelet thrombotic and non-thrombotic functions.
Assuntos
Plaquetas/metabolismo , Regulação da Expressão Gênica , Infarto do Miocárdio/genética , Transcriptoma , Idoso , Idoso de 80 Anos ou mais , Eletrocardiografia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/diagnóstico , Ativação Plaquetária , Agregação Plaquetária , Testes de Função Plaquetária , Fatores de Risco , Análise de Sequência de RNARESUMO
Glucocorticoids remain the cornerstone of treatment for inflammatory conditions, but their utility is limited by a plethora of side effects. One of the key goals of immunotherapy across medical disciplines is to minimize patients' glucocorticoid use. Increasing evidence suggests that variations in the adaptive immune response play a critical role in defining the dose of glucocorticoids required to control an individual's disease, and Th17 cells are strong candidate drivers for nonresponsiveness [also called steroid resistance (SR)]. Here we use gene-expression profiling to further characterize the SR phenotype in T cells and show that Th17 cells generated from both SR and steroid-sensitive individuals exhibit restricted genome-wide responses to glucocorticoids in vitro, and that this is independent of glucocorticoid receptor translocation or isoform expression. In addition, we demonstrate, both in transgenic murine T cells in vitro and in an in vivo murine model of autoimmunity, that Th17 cells are reciprocally sensitive to suppression with the calcineurin inhibitor, cyclosporine A. This result was replicated in human Th17 cells in vitro, which were found to have a conversely large genome-wide shift in response to cyclosporine A. These observations suggest that the clinical efficacy of cyclosporine A in the treatment of SR diseases may be because of its selective attenuation of Th17 cells, and also that novel therapeutics, which target either Th17 cells themselves or the effector memory T-helper cell population from which they are derived, would be strong candidates for drug development in the context of SR inflammation.
Assuntos
Ciclosporina/química , Glucocorticoides/química , Células Th17/citologia , Animais , Autoimunidade , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Calcineurina/química , Inibidores de Calcineurina/química , Núcleo Celular/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Humanos , Inflamação , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos , Camundongos Transgênicos , Fenótipo , Esteroides/químicaRESUMO
The pathophysiology of severe aplastic anemia (SAA) is immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). Most patients respond to immunosuppressive therapies, but a minority transform to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), frequently associated with monosomy 7 (-7). Thirteen SAA patients were analyzed for acquired mutations in myeloid cells at the time of evolution to -7, and all had a dominant HSPC clone bearing specific acquired mutations. However, mutations in genes associated with MDS/AML were present in only 4 cases. Patients who evolved to MDS and AML showed marked progressive telomere attrition before the emergence of -7. Single telomere length analysis confirmed accumulation of short telomere fragments of individual chromosomes. Our results indicate that accelerated telomere attrition in the setting of a decreased HSPC pool is characteristic of early myeloid oncogenesis, specifically chromosome 7 loss, in MDS/AML after SAA, and provides a possible mechanism for development of aneuploidy.
Assuntos
Anemia Aplástica/genética , Células-Tronco Hematopoéticas , Homeostase do Telômero , Anemia Aplástica/metabolismo , Anemia Aplástica/patologia , Deleção Cromossômica , Cromossomos Humanos Par 7/genética , Cromossomos Humanos Par 7/metabolismo , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologiaRESUMO
The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for malaria. Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown. Here we show that knocking out the P. falciparum variant-silencing SET gene (here termed PfSETvs), which encodes an orthologue of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual infected red blood cells. PfSETvs-dependent H3K36me3 is present along the entire gene body, including the transcription start site, to silence var genes. With low occupancy of PfSETvs at both the transcription start site of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncover a previously unknown role of PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which orthologues of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine.
Assuntos
Inativação Gênica , Histonas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , Fatores de Virulência/genética , Proteínas de Ligação a DNA , Proteínas de Drosophila , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Genes de Protozoários/genética , Histonas/química , Íntrons/genética , Lisina/metabolismo , Vacinas Antimaláricas/genética , Metilação , Plasmodium falciparum/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/genética , RNA Longo não Codificante/genética , Fatores de Transcrição , Sítio de Iniciação de Transcrição , Virulência/genéticaRESUMO
Multiple myeloma (MM) is characterized by the malignant expansion of differentiated plasma cells. Although many chemotherapeutic agents display cytotoxic activity toward MM cells, patients inevitably succumb to their disease because the tumor cells become resistant to the anticancer drugs. The cancer stem cell hypothesis postulates that a small subpopulation of chemotherapy-resistant cancer cells is responsible for propagation of the tumor. Herein we report that efflux of the pluripotent stem cell dye CDy1 identifies a subpopulation in MM cell lines characterized by increased expression of P-glycoprotein, a member of the ABC (ATP-binding cassette) superfamily of transporters encoded by ABCB1. We also demonstrate that ABCB1-overexpressing MM cells are resistant to the second-generation proteasome inhibitor carfilzomib that recently received accelerated approval for the treatment of therapy-refractive MM by the U.S. Food and Drug Administration. Moreover, increased resistance to carfilzomib in sensitive MM cells following drug selection was associated with upregulation of ABCB1 cell-surface expression which correlated with increased transporter activity as measured by CDy1 efflux. We further show that chemosensitization of MM cells to carfilzomib could be achieved in vitro by cotreatment with vismodegib, a hedgehog pathway antagonist which is currently in MM clinical trials. CDy1 efflux may therefore be a useful assay to determine whether high expression of ABCB1 is predictive of poor clinical responses in MM patients treated with carfilzomib. Our data also suggest that inclusion of vismodegib might be a potential strategy to reverse ABCB1-mediated drug resistance should it occur.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antracenos , Antineoplásicos/farmacologia , Morfolinas , Mieloma Múltiplo/patologia , Células-Tronco Neoplásicas/patologia , Oligopeptídeos/farmacologia , Células-Tronco Pluripotentes/patologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Anilidas/farmacologia , Transporte Biológico , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Corantes Fluorescentes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Plasmócitos/efeitos dos fármacos , Plasmócitos/metabolismo , Plasmócitos/patologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Piridinas/farmacologiaRESUMO
This study describes a unique function of taurocholate in bile canalicular formation involving signaling through a cAMP-Epac-MEK-Rap1-LKB1-AMPK pathway. In rat hepatocyte sandwich cultures, polarization was manifested by sequential progression of bile canaliculi from small structures to a fully branched network. Taurocholate accelerated canalicular network formation and concomitantly increased cAMP, which were prevented by adenyl cyclase inhibitor. The cAMP-dependent PKA inhibitor did not prevent the taurocholate effect. In contrast, activation of Epac, another cAMP downstream kinase, accelerated canalicular network formation similar to the effect of taurocholate. Inhibition of Epac downstream targets, Rap1 and MEK, blocked the taurocholate effect. Taurocholate rapidly activated MEK, LKB1, and AMPK, which were prevented by inhibition of adenyl cyclase or MEK. Our previous study showed that activated-LKB1 and AMPK participate in canalicular network formation. Linkage between bile acid synthesis, hepatocyte polarization, and regulation of energy metabolism is likely important in normal hepatocyte development and disease.
Assuntos
Ácidos e Sais Biliares/farmacologia , Polaridade Celular/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Quinases Proteína-Quinases Ativadas por AMP , Adenilato Quinase/antagonistas & inibidores , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Animais , Western Blotting , Células Cultivadas , Ácido Quenodesoxicólico/farmacologia , Colagogos e Coleréticos/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Iminas/farmacologia , Microscopia Confocal , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Ácido Taurocólico/farmacologia , Ácido Ursodesoxicólico/farmacologia , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
AMP-activated protein kinase (AMPK), a cellular metabolic sensor, is essential in energy regulation and metabolism. Hepatocyte polarization during liver development and regeneration parallels increased metabolism. The current study investigates the effects of AMPK and its upstream activator LKB1 on polarity and bile canalicular network formation and maintenance in collagen sandwich cultures of rat hepatocytes. Immunostaining for the apical protein ABCB1 and the tight junction marker occludin demonstrated that canalicular network formation is sequential and is associated with activation of AMPK and LKB1. AMPK and LKB1 activators accelerated canalicular network formation. Inhibition of AMPK or LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs blocked canalicular network formation. AICAR and 2-deoxyglucose, which activate AMPK, circumvented the inhibitory effect of kinase-dead LKB1 on canalicular formation, indicating that AMPK directly affects canalicular network formation. After the canalicular network was formed, inhibition of AMPK and LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs resulted in loss of canalicular network, indicating that AMPK and LKB1 also participate in network maintenance. In addition, activation of AMPK and LKB1 prevented low-Ca(2+)-mediated disruption of the canalicular network and tight junctions. These studies reveal that AMPK and its upstream kinase, LKB1, regulate canalicular network formation and maintenance.
Assuntos
Canalículos Biliares/metabolismo , Hepatócitos/metabolismo , Proteínas Mutantes/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Canalículos Biliares/crescimento & desenvolvimento , Polaridade Celular/genética , Células Cultivadas , Clonagem Molecular , Ativação Enzimática/genética , Hepatócitos/patologia , Masculino , Proteínas Mutantes/genética , Técnicas de Cultura de Órgãos , Organogênese/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Sprague-DawleyRESUMO
Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC) is a multisystem disorder associated with abnormalities in polarized liver and kidney cells. Mutations in VPS33B account for most cases of ARC. We identified mutations in VIPAR (also called C14ORF133) in individuals with ARC without VPS33B defects. We show that VIPAR forms a functional complex with VPS33B that interacts with RAB11A. Knockdown of vipar in zebrafish resulted in biliary excretion and E-cadherin defects similar to those in individuals with ARC. Vipar- and Vps33b-deficient mouse inner medullary collecting duct (mIMDC-3) cells expressed membrane proteins abnormally and had structural and functional tight junction defects. Abnormal Ceacam5 expression was due to mis-sorting toward lysosomal degradation, but reduced E-cadherin levels were associated with transcriptional downregulation. The VPS33B-VIPAR complex thus has diverse functions in the pathways regulating apical-basolateral polarity in the liver and kidney.
Assuntos
Artrogripose/genética , Proteínas de Transporte/genética , Colestase/genética , Nefropatias/genética , Proteínas de Membrana/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Polaridade Celular , Epitélio/fisiologia , Humanos , Camundongos , Mutação , Fenótipo , Síndrome , Junções Íntimas/patologia , Proteínas de Transporte Vesicular , Peixe-ZebraRESUMO
Arthrogryposis, Renal dysfunction and Cholestasis (ARC) syndrome is a multi-system autosomal recessive disorder caused by germline mutations in VPS33B. The detection of germline VPS33B mutations removes the need for diagnostic organ biopsies (these carry a>50% risk of life-threatening haemorrhage due to platelet dysfunction); however, VPS33B mutations are not detectable in approximately 25% of patients. In order further to define the molecular basis of ARC we performed mutation analysis and mRNA and protein studies in patients with a clinical diagnosis of ARC. Here we report novel mutations in VPS33B in patients from Eastern Europe and South East Asia. One of the mutations was present in 7 unrelated Korean patients. Reduced expression of VPS33B and cellular phenotype was detected in fibroblasts from patients clinically diagnosed with ARC with and without known VPS33B mutations. One mutation-negative patient was found to have normal mRNA and protein levels. This patient's clinical condition improved and he is alive at the age of 2.5 years. Thus we show that all patients with a classical clinical course of ARC had decreased expression of VPS33B whereas normal VPS33B expression was associated with good prognosis despite initial diagnosis of ARC.
Assuntos
Artrogripose/complicações , Artrogripose/diagnóstico , Colestase/complicações , Colestase/diagnóstico , Nefropatias/complicações , Nefropatias/diagnóstico , Artrogripose/etnologia , Pré-Escolar , Colestase/etnologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Lactente , Nefropatias/etnologia , Masculino , Mutação/genética , Síndrome , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
UNLABELLED: Cyclic adenosine monophosphate (cAMP) stimulates hepatic bile acid uptake by translocating sodium-taurocholate (TC) cotransporting polypeptide (Ntcp) from an endosomal compartment to the plasma membrane. Rab4 is associated with early endosomes and involved in vesicular trafficking. This study was designed to determine the role of Rab4 in cAMP-induced TC uptake and Ntcp translocation. HuH-Ntcp cells transiently transfected with empty vector, guanosine triphosphate (GTP) locked dominant active Rab4 (Rab4(GTP)), or guanosine diphosphate (GDP) locked dominant inactive Rab4 (Rab4(GDP)) were used to study the role of Rab4. Neither Rab4(GTP) nor Rab4(GDP) affected either basal TC uptake or plasma membrane Ntcp level. However, cAMP-induced increases in TC uptake and Ntcp translocation were enhanced by Rab4(GTP) and inhibited by Rab4(GDP). In addition, cAMP increased GTP binding to endogenous Rab4 in a time-dependent, but phosphoinositide-3-kinase-independent manner. CONCLUSION: Taken together, these results suggest that cAMP-mediated phosphoinositide-3-kinase-independent activation of Rab4 facilitates Ntcp translocation in HuH-Ntcp cells.