RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes injury to multiple organ systems, including the brain. SARS-CoV-2's neuropathological mechanisms may include systemic inflammation and hypoxia, as well as direct cell damage resulting from viral infections of neurons and glia. How the virus directly causes injury to brain cells, acutely and over the long term, is not well understood. In order to gain insight into this process, we studied the neuropathological effects of open reading frame 3a (ORF3a), a SARS-CoV-2 accessory protein that is a key pathological factor of the virus. Forced ORF3a brain expression in mice caused the rapid onset of neurological impairment, neurodegeneration, and neuroinflammation-key neuropathological features found in coronavirus disease (COVID-19, which is caused by SARS-CoV-2 infection). Furthermore, ORF3a expression blocked autophagy progression in the brain and caused the neuronal accumulation of α-synuclein and glycosphingolipids, all of which are linked to neurodegenerative disease. Studies with ORF3-expressing HeLa cells confirmed that ORF3a disrupted the autophagy-lysosomal pathway and blocked glycosphingolipid degradation, resulting in their accumulation. These findings indicate that, in the event of neuroinvasion by SARS-CoV-2, ORF3a expression in brain cells may drive neuropathogenesis and be an important mediator of both short- and long-term neurological manifestations of COVID-19.
Assuntos
COVID-19 , Doenças Neurodegenerativas , Animais , Humanos , Camundongos , Autofagia , Encéfalo/patologia , COVID-19/patologia , Células HeLa , Homeostase , Lisossomos , Doenças Neurodegenerativas/patologia , Fases de Leitura Aberta , SARS-CoV-2 , EsfingolipídeosRESUMO
GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes lysosomal ß-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residual ß-galactosidase activity primarily determining the clinical course. Glb1 null mouse models, which completely lack ß-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generated Glb1/Neu3 double KO (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia than Glb1 KO mice. Glb1/Neu3 DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared with Glb1 KO mice, indicating that NEU3 mediated GM1 ganglioside to GA1 glycolipid conversion in Glb1 KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced in Glb1/Neu3 DKO mice compared with Glb1 KO mice. Mouse NEU3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight NEU3's role in ameliorating the consequences of Glb1 deletion in mice, provide insights into NEU3's differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients.
Assuntos
Gangliosidose GM1 , Animais , Humanos , Camundongos , beta-Galactosidase/genética , beta-Galactosidase/metabolismo , beta-Galactosidase/uso terapêutico , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M1)/uso terapêutico , Gangliosidose GM1/genética , Glicolipídeos , Neuraminidase/genética , Neuraminidase/uso terapêuticoRESUMO
Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes. In some cases, large amounts of specific sphingolipids must be synthesized for specialized physiological functions, such as during axon myelination. How sphingolipid synthesis is regulated to fulfill these physiological requirements is not known. To identify genes that positively regulate membrane sphingolipid levels, here we employed a genome-wide CRISPR/Cas9 loss-of-function screen in HeLa cells using selection for resistance to Shiga toxin, which uses a plasma membrane-associated glycosphingolipid, globotriaosylceramide (Gb3), for its uptake. The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it also identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis. AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis. AHR knockout HeLa cells exhibited significantly reduced levels of cell-surface Gb3, and both AHR knockout HeLa cells and tissues from Ahr knockout mice displayed decreased sphingolipid content as well as significantly reduced expression of several key genes in the sphingolipid biosynthetic pathway. The sciatic nerve of Ahr knockout mice exhibited both reduced ceramide content and reduced myelin thickness. These results indicate that AHR up-regulates sphingolipid levels and is important for full axon myelination, which requires elevated levels of membrane sphingolipids.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Resistência à Doença/genética , Globosídeos/genética , Receptores de Hidrocarboneto Arílico/genética , Serina C-Palmitoiltransferase/genética , Esfingolipídeos/biossíntese , Triexosilceramidas/genética , Animais , Sistemas CRISPR-Cas/genética , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Genoma Humano/genética , Células HeLa , Humanos , Metabolismo dos Lipídeos/genética , Lipídeos/biossíntese , Lipídeos/genética , Camundongos , Camundongos Knockout , Toxina Shiga/farmacologia , Transdução de Sinais/genética , Esfingolipídeos/genéticaRESUMO
Here we investigated in primary human erythroid tissues a downstream element of the heterochronic let-7 miRNA pathway, the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), for its potential to affect the hemoglobin profiles in human erythroblasts. Comparison of adult bone marrow to fetal liver lysates demonstrated developmental silencing in IGF2BP1. Erythroid-specific overexpression of IGF2BP1 caused a nearly complete and pancellular reversal of the adult pattern of hemoglobin expression toward a more fetal-like phenotype. The reprogramming of hemoglobin expression was achieved at the transcriptional level by increased gamma-globin combined with decreased beta-globin transcripts resulting in gamma-globin rising to 90% of total beta-like mRNA. Delta-globin mRNA was reduced to barely detectable levels. Alpha-globin levels were not significantly changed. Fetal hemoglobin achieved levels of 68.6 ± 3.9% in the IGF2BP1 overexpression samples compared with 5.0 ± 1.8% in donor matched transduction controls. In part, these changes were mediated by reduced protein expression of the transcription factor BCL11A. mRNA stability and polysome studies suggest IGF2BP1 mediates posttranscriptional loss of BCL11A. These results suggest a mechanism for chronoregulation of fetal and adult hemoglobin expression in humans.
Assuntos
Proteínas de Transporte/metabolismo , Eritroblastos/metabolismo , Hemoglobina Fetal/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Medula Óssea/metabolismo , Células HEK293 , Proteína HMGA2/metabolismo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fígado/embriologia , Fenótipo , RNA Mensageiro/metabolismo , Proteínas Repressoras , Globinas beta/metabolismo , gama-Globinas/metabolismoRESUMO
BACKGROUND: In humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19-40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts. METHODS: The expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins. RESULTS: The expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged. CONCLUSIONS: Overall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , Adulto , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Proliferação de Células/genética , Células Cultivadas , Hemoglobina Fetal , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , MicroRNAs/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras , Reticulócitos/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismoRESUMO
Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and ß-thalassemia. Transcription of ß-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult ß-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on ß-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult ß-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of ß-hemoglobinopathies.
Assuntos
Hemoglobina Fetal/biossíntese , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Região de Controle de Locus Gênico , gama-Globinas/genética , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Diferenciação Celular , Proteínas de Ligação a DNA/sangue , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Eritropoese , Antígenos de Histocompatibilidade , Humanos , Técnicas In Vitro , Proteínas com Domínio LIM/sangue , Modelos Biológicos , Regiões Promotoras Genéticas , Quinazolinas/farmacologia , Fatores de Transcrição/sangue , Talassemia beta/sangue , Talassemia beta/tratamento farmacológico , Talassemia beta/genéticaRESUMO
Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and ß-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of γ-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Eritroblastos/citologia , Eritrócitos/citologia , Hemoglobina Fetal/metabolismo , Regulação da Expressão Gênica , Antígenos CD34/metabolismo , Anidrase Carbônica I/metabolismo , Técnicas de Cultura de Células , Sangue Fetal/citologia , Hemoglobina A/metabolismo , Humanos , MicroRNAs/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Fenótipo , Proteínas de Ligação a RNARESUMO
In thalassemia, deficient globin-chain production during erythropoiesis results in anemia. Thalassemia may be further complicated by iron overload (frequently exacerbated by blood transfusion), which induces numerous endocrine diseases, hepatic cirrhosis, cardiac failure and even death. Accumulation of iron in the absence of blood transfusions may result from inappropriate suppression of the iron-regulating peptide hepcidin by an erythropoietic mechanism. To test this hypothesis, we examined erythroblast transcriptome profiles from 15 healthy, nonthalassemic donors. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-beta superfamily, showed increased expression and secretion during erythroblast maturation. Healthy volunteers had mean GDF15 serum concentrations of 450 +/- 50 pg/ml. In comparison, individuals with beta-thalassemia syndromes had elevated GDF15 serum levels (mean 66,000 +/- 9,600 pg/ml; range 4,800-248,000 pg/ml; P < 0.05) that were positively correlated with the levels of soluble transferrin receptor, erythropoietin and ferritin. Serum from thalassemia patients suppressed hepcidin mRNA expression in primary human hepatocytes, and depletion of GDF15 reversed hepcidin suppression. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Citocinas/sangue , Regulação da Expressão Gênica , Talassemia/sangue , Talassemia/genética , Perfilação da Expressão Gênica , Fator 15 de Diferenciação de Crescimento , Hepcidinas , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Valores de Referência , Transcrição GênicaRESUMO
The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the ß-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the ß-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the (A)γ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing ß-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and ß-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.
Assuntos
Proteínas de Ligação a DNA/genética , Células Eritroides/metabolismo , Proteínas com Domínio LIM/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , gama-Globinas/genética , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Eritroides/citologia , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Humanos , Células K562 , Proteínas com Domínio LIM/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA não Traduzido/genética , Proteínas Repressoras/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Proteínas Supressoras de Tumor/metabolismo , gama-Globinas/metabolismoRESUMO
In thalassemia and other iron loading anemias, ineffective erythropoiesis and erythroid signaling molecules are thought to cause inappropriate suppression of a small peptide produced by hepatocytes named hepcidin. Previously, it was reported that the erythrokine GDF15 is expressed at very high levels in thalassemia and suppresses hepcidin expression. In this study, erythroblast expression of a second molecule named twisted gastrulation (TWSG1) was explored as a potential erythroid regulator of hepcidin. Transcriptome analyses suggest TWSG1 is produced during the earlier stages of erythropoiesis. Hepcidin suppression assays demonstrated inhibition by TWSG1 as measured by quantitative polymerase chain reaction (PCR) in dosed assays (1-1000 ng/mL TWSG1). In human cells, TWSG1 suppressed hepcidin indirectly by inhibiting the signaling effects and associated hepcidin up-regulation by bone morphogenic proteins 2 and 4 (BMP2/BMP4). In murine hepatocytes, hepcidin expression was inhibited by murine Twsg1 in the absence of additional BMP. In vivo studies of Twsg1 expression were performed in healthy and thalassemic mice. Twsg1 expression was significantly increased in the spleen, bone marrow, and liver of the thalassemic animals. These data demonstrate that twisted gastrulation protein interferes with BMP-mediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes.
Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Citocinas/fisiologia , Eritropoese/fisiologia , Proteínas/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteína Morfogenética Óssea 2/fisiologia , Proteína Morfogenética Óssea 4/fisiologia , Citocinas/genética , Eritropoese/genética , Feminino , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/fisiologia , Hepatócitos/citologia , Hepatócitos/fisiologia , Hepcidinas , Homeostase , Humanos , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas/genética , Proteínas Smad/fisiologia , Talassemia/sangue , Talassemia/genética , Talassemia/patologia , Talassemia/fisiopatologiaRESUMO
Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34(+) cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.
Assuntos
Citocinas/metabolismo , Células Eritroides/metabolismo , Hemoglobina Fetal/biossíntese , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Adulto , Antígenos CD34 , Células Cultivadas , Células Eritroides/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hemoglobinopatias/metabolismo , Humanos , RNA Polimerase II/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
BACKGROUND: Low serum hepcidin levels provide a physiologic response to iron demand in patients with iron deficiency (ID). Based on a discovery of suppressed hepcidin expression by a cytokine named growth differentiation factor 15 (GDF15), it was hypothesized that GDF15 may suppress hepcidin expression in humans with ID due to blood loss. STUDY DESIGN AND METHODS: To test this hypothesis, GDF15 and hepcidin levels were measured in peripheral blood from subjects with iron-deficient erythropoiesis before and after iron supplementation. RESULTS: Iron variables and hepcidin levels were significantly suppressed in iron-deficient blood donors compared to healthy volunteers. However, ID was not associated with elevated serum levels of GDF15. Instead, iron-deficient subjects' GDF15 levels were slightly lower than those measured in the control group of subjects (307 +/- 90 and 386 +/- 104 pg/mL, respectively). Additionally, GDF15 levels were not significantly altered by iron repletion. CONCLUSIONS: ID due to blood loss is not associated with a significant change in serum levels of GDF15.
Assuntos
Doadores de Sangue , Fator 15 de Diferenciação de Crescimento/sangue , Deficiências de Ferro , Peptídeos Catiônicos Antimicrobianos/análise , Ferritinas/sangue , Hepcidinas , Humanos , Transferrina/análiseRESUMO
Sickle cell disease (SCD) and ß-thalassemia are caused by structural abnormality or inadequate production of adult hemoglobin (HbA, α2ß2), respectively. Individuals with either disorder are asymptomatic before birth because fetal hemoglobin (HbF, α2γ2) is unaffected. Thus, reversal of the switch from HbF to HbA could reduce or even prevent symptoms these disorders. In this study, we show that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is one factor that could accomplish this goal. IGF2BP1 is a fetal factor that undergoes a transcriptional switch consistent with the transition from HbF to HbA. Lentivirus delivery of IGF2BP1 to CD34+ cells of healthy adult donors reversed hemoglobin production toward the fetal type in culture-differentiated erythroid cells. Analogous studies using patient-derived CD34+ cells revealed that IGF2BP1-dependent HbF induction could ameliorate the chain imbalance in ß-thalassemia or potently suppress expression of sickle ß-globin in SCD. In all cases, fetal γ-globin mRNA increased and adult ß-globin decreased due, in part, to formation of contacts between the locus control region (LCR) and γ-globin genes. We conclude that expression of IGF2BP1 in adult erythroid cells has the potential to maximize HbF expression in patients with severe ß-hemoglobin disorders by reversing the developmental γ- to ß-globin switch.
RESUMO
In vivo, inhibition of fetal hemoglobin (HbF) expression in humans around the time of birth causes the clinical manifestation of sickle cell and beta-thalassemia syndromes. Inhibition of HbF among cultured cells was recently described by the adenosine derivative molecule named SQ22536. Here, a primary cell culture model was utilized to further explore the inhibition of HbF by adenosine derivative molecules. SQ22536 demonstrated down-regulation of growth and HbF expression among erythroblasts cultured from fetal and adult human blood. The effects upon HbF were noted in a majority of cells, and quantitative PCR analysis demonstrated a transcriptional mechanism. Screening assays demonstrated that two additional molecules named 5'-deoxy adenosine and 2',3'-dideoxy adenosine had effects on HbF comparable to SQ22536. Other adenosine derivative molecules, adenosine receptor binding ligands, and cAMP-signaling regulators failed to inhibit HbF in matched cultures. These results suggest that structurally related ribofuranose-substituted adenosine analogues act through an unknown mechanism to inhibit HbF expression in fetal and adult human erythroblasts.
Assuntos
Adenosina/farmacologia , Eritroblastos/citologia , Eritroblastos/efeitos dos fármacos , Hemoglobina Fetal/biossíntese , Adenina/análogos & derivados , Adenina/química , Adenina/farmacologia , Adenosina/química , Adulto , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/metabolismo , Desoxiadenosinas/química , Desoxiadenosinas/farmacologia , Didesoxiadenosina/química , Didesoxiadenosina/farmacologia , Eritropoetina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Globinas/genética , Globinas/metabolismo , Humanos , Cinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Células-Tronco/farmacologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
BACKGROUND: MicroRNAs are approximately 22nt-long small non-coding RNAs that negatively regulate protein expression through mRNA degradation or translational repression in eukaryotic cells. Based upon their importance in regulating development and terminal differentiation in model systems, erythrocyte microRNA profiles were examined at birth and in adults to determine if changes in their abundance coincide with the developmental phenomenon of hemoglobin switching. METHODS: Expression profiling of microRNA was performed using total RNA from four adult peripheral blood samples compared to four cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNAs were hybridized to custom spotted arrays containing 474 human microRNA species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines provided a hybridization reference for all samples to generate microRNA abundance profile for each sample. RESULTS: Among 206 detected miRNAs, 79% of the microRNAs were present at equivalent levels in both cord and adult cells. By comparison, 37 microRNAs were up-regulated and 4 microRNAs were down-regulated in adult erythroid cells (fold change > 2; p < 0.01). Among the up-regulated subset, the let-7 miRNA family consistently demonstrated increased abundance in the adult samples by array-based analyses that were confirmed by quantitative PCR (4.5 to 18.4 fold increases in 6 of 8 let-7 miRNA). Profiling studies of messenger RNA (mRNA) in these cells additionally demonstrated down-regulation of ten let-7 target genes in the adult cells. CONCLUSION: These data suggest that a consistent pattern of up-regulation among let-7 miRNA in circulating erythroid cells occurs in association with hemoglobin switching during the fetal-to-adult developmental transition in humans.
Assuntos
Células Eritroides , MicroRNAs/metabolismo , Reticulócitos/fisiologia , Adulto , Animais , Sangue Fetal/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hemoglobinas/genética , Humanos , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos TestesRESUMO
Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.
Assuntos
Proteínas de Membrana/genética , Bainha de Mielina/genética , Esfingolipídeos/genética , Animais , Ceramidas/genética , Células HeLa , Humanos , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Camundongos , Camundongos Knockout , Bainha de Mielina/metabolismo , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Transdução de Sinais/genética , Esfingolipídeos/biossínteseRESUMO
RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133 arrays were hybridized with probe generated from the reticulocyte total RNA (blood obtained from 14 umbilical cords and 14 healthy adult humans). Among the cord and adult reticulocyte profiles, 698 probe sets (488 named genes) were detected in each of the 28 samples. Among the highly expressed genes, promoter analyses revealed a subset of transcription factor binding motifs encoded at higher than expected frequencies including the hypoxia-related arylhydrocarbon receptor repressor family. Over 100 probe sets demonstrated differential expression between the cord and adult reticulocyte samples. For verification, the array expression patterns for 21 genes were confirmed by real-time PCR (correlation coefficient 0.98). Only four transcripts (MAP17, FLJ32009, ARRB2, and FLJ27365) were identified as being upregulated in the adult blood transcriptome. Further analysis revealed that the lipid-regulating protein MAP17 was present in the membrane fraction of adult erythrocytes, but not detected in cord blood erythrocytes. Combined with other clinical and experimental data, these reticulocyte transcriptome profiles should be useful to better understand the molecular bases of terminal erythroid differentiation, hemoglobin switching, iron metabolism and malarial pathogenesis.
Assuntos
RNA Mensageiro/genética , Reticulócitos/metabolismo , Separação Celular , Biologia Computacional , Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Sondas RNARESUMO
Hembase (http://hembase.niddk.nih.gov) is an integrated browser and genome portal designed for web-based examination of the human erythroid transcriptome. To date, Hembase contains 15,752 entries from erythroblast Expressed Sequenced Tags (ESTs) and 380 referenced genes relevant for erythropoiesis. The database is organized to provide a cytogenetic band position, a unique name as well as a concise annotation for each entry. Search queries may be performed by name, keyword or cytogenetic location. Search results are linked to primary sequence data and three major human genome browsers for access to information considered current at the time of each search. Hembase provides interested scientists and clinical hematologists with a genome-based approach toward the study of erythroid biology.
Assuntos
Bases de Dados Genéticas , Eritrócitos/metabolismo , Eritropoese/genética , Genômica , Hematologia , Biologia Computacional , Citogenética , Etiquetas de Sequências Expressas , Genoma Humano , Humanos , Armazenamento e Recuperação da Informação , Internet , Transcrição Gênica/genéticaRESUMO
Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with beta-hemoglobin disorders. Previous studies showed that let-7 microRNAs (miRNAs) are highly regulated in erythroid cells during the fetal-to-adult developmental transition, and that targeting let-7 mediated the up-regulation of HbF to greater than 30% of the total globin levels in human adult cultured erythroblasts. HMGA2 is a member of the high-mobility group A family of proteins and a validated target of the let-7 family of miRNAs. Here we investigate whether expression of HMGA2 directly regulates fetal hemoglobin in adult erythroblasts. Let-7 resistant HMGA2 expression was studied after lentiviral transduction of CD34(+) cells. The transgene was regulated by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2-OE). HMGA2-OE caused significant increases in gamma-globin mRNA expression and HbF to around 16% of the total hemoglobin levels compared to matched control transductions. Interestingly, no significant changes in KLF1, SOX6, GATA1, ZBTB7A and BCL11A mRNA levels were observed. Overall, our data suggest that expression of HMGA2, a downstream target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in adult human erythroblasts.