RESUMO
BACKGROUND: Patients with non-transfusion-dependent thalassaemia (NTDT), although they do not require regular blood transfusions for survival, can still accrue a heavy burden of comorbidities. No approved disease-modifying therapies exist for these patients. We aimed to investigate the safety and efficacy of mitapivat (Agios Pharmaceuticals, Cambridge, MA, USA), a pyruvate kinase activator, in adults with non-transfusion-dependent (NTD) α-thalassaemia or NTD ß-thalassaemia. METHODS: In this open-label, multicentre, phase 2 study, patients were recruited from four academic clinical study sites in Oakland, CA, and Boston, MA, USA; Toronto, ON, Canada; and London, UK. Patients were eligible if they were aged 18 years or older, with NTDT (including ß-thalassaemia with or without α-globin gene mutations, haemoglobin E ß-thalassaemia, or α-thalassaemia), and a baseline haemoglobin concentration of 10·0 g/dL or lower. During a 24-week core period, mitapivat was administered orally at 50 mg twice daily for the first 6 weeks followed by an escalation to 100 mg twice daily for 18 weeks thereafter. The primary endpoint was haemoglobin response (a ≥1·0 g/dL increase in haemoglobin concentration from baseline at one or more assessments between weeks 4 and 12). Efficacy and safety were assessed in the full analysis set (ie, all patients who received at least one dose of study drug). This study is registered with ClinicalTrials.gov, NCT03692052, and is closed to accrual. FINDINGS: Between Dec 28, 2018, and Feb 6, 2020, 27 patients were screened, of whom 20 were enrolled (15 [75%] with ß-thalassaemia and five [25%] with α-thalassaemia) and received mitapivat. The median age of patients was 44 years (IQR 35-56), 15 (75%) of 20 patients were female, five (25%) were male, and ten (50%) identified as Asian. 16 (80% [90% CI 60-93]) of 20 patients had a haemoglobin response (p<0·0001), five (100%) of five with α-thalassaemia and 11 (73%) of 15 with ß-thalassaemia. 17 (85%) patients had a treatment-emergent adverse event, and 13 had a treatment-emergent event that was considered to be treatment related. One serious treatment-emergent adverse event occurred (grade 3 renal impairment), which was considered unrelated to study drug, resulting in discontinuation of treatment. The most commonly reported treatment-emergent adverse events were initial insomnia (ten [50%] patients), dizziness (six [30%]), and headache (five [25%]). No patients died during the 24-week core period. INTERPRETATION: These efficacy and safety results support the continued investigation of mitapivat for the treatment of both α-thalassaemia and ß-thalassaemia. FUNDING: Agios Pharmaceuticals.
Assuntos
Piperazinas , Quinolinas , Talassemia alfa , Talassemia beta , Adulto , Feminino , Hemoglobinas , Humanos , Masculino , Pessoa de Meia-Idade , Piperazinas/efeitos adversos , Piruvato Quinase , Quinolinas/efeitos adversos , Talassemia alfa/tratamento farmacológico , Talassemia beta/tratamento farmacológicoRESUMO
Polymerization of deoxygenated hemoglobin S underlies the pathophysiology of sickle cell disease (SCD). In activating red blood cell pyruvate kinase and glycolysis, mitapivat (AG-348) increases adenosine triphosphate (ATP) levels and decreases the 2,3-diphosphoglycerate (2,3-DPG) concentration, an upstream precursor in glycolysis. Both changes have therapeutic potential for patients with SCD. Here, we evaluated the safety and tolerability of multiple ascending doses of mitapivat in adults with SCD with no recent blood transfusions or changes in hydroxyurea or l-glutamine therapy. Seventeen subjects were enrolled; 1 subject was withdrawn shortly after starting the study. Sixteen subjects completed 3 ascending dose levels of mitapivat (5, 20, and 50 mg, twice daily [BID]) for 2 weeks each; following a protocol amendment, the dose was escalated to 100 mg BID in 9 subjects. Mitapivat was well tolerated at all dose levels, with the most common treatment-emergent adverse events (AEs) being insomnia, headache, and hypertension. Six serious AEs (SAEs) included 4 vaso-occlusive crises (VOCs), non-VOC-related shoulder pain, and a preexisting pulmonary embolism. Two VOCs occurred during drug taper and were possibly drug related; no other SAEs were drug related. Mean hemoglobin increase at the 50 mg BID dose level was 1.2 g/dL, with 9 of 16 (56.3%) patients achieving a hemoglobin response of a ≥1 g/dL increase compared with baseline. Mean reductions in hemolytic markers and dose-dependent decreases in 2,3-DPG and increases in ATP were also observed. This study provides proof of concept that mitapivat has disease-modifying potential in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.
Assuntos
Anemia Falciforme , Piruvato Quinase , Adulto , Humanos , Ácido Pirúvico , 2,3-Difosfoglicerato , Anemia Falciforme/tratamento farmacológico , Hemoglobinas , Trifosfato de AdenosinaRESUMO
Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels.
Assuntos
Anemia Falciforme , 2,3-Difosfoglicerato/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Modelos Animais de Doenças , Eritrócitos/metabolismo , Hemoglobina Falciforme/metabolismo , Hemoglobinas/análise , Humanos , Camundongos , Mitocôndrias/metabolismo , Estresse Oxidativo , Piperazinas , QuinolinasRESUMO
Anemia in ß-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on ß-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for ß-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in ß-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing ß-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with ß-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for ß-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.
Assuntos
Ativadores de Enzimas/farmacologia , Hemólise/efeitos dos fármacos , Piperazinas/farmacologia , Piruvato Quinase/metabolismo , Quinolinas/farmacologia , Talassemia beta/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Transgênicos , Talassemia beta/enzimologia , Talassemia beta/genéticaAssuntos
Anemia Falciforme/enzimologia , Eritrócitos Anormais/enzimologia , Terapia de Alvo Molecular , Piperazinas/farmacologia , Piruvato Quinase/deficiência , Quinolinas/farmacologia , 2,3-Difosfoglicerato/sangue , Adolescente , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Forma Celular/efeitos dos fármacos , Criança , Pré-Escolar , Eritrócitos Anormais/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Piperazinas/uso terapêutico , Estabilidade Proteica , Piruvato Quinase/sangue , Piruvato Quinase/química , Quinolinas/uso terapêutico , Adulto JovemRESUMO
Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red cell (RBC) glycolysis, causing changes in metabolism including a deficiency in ATP. This affects red cell homeostasis, promoting premature removal of RBCs from the circulation. In this study we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBCs and erythroid precursors from PK-deficient patients. In 15 patients ex vivo treatment with AG-348 resulted in increased enzymatic activity in all patient cells after 24 hours (mean increase 1.8-fold, range 1.2-3.4). ATP levels increased (mean increase 1.5-fold, range 1.0-2.2) similar to control cells (mean increase 1.6-fold, range, 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBCs. Ex vivo treatment with AG-348 increased residual activity 1.4 to >10-fold than residual activity of vehicle-treated samples. Protein analyses suggests that a sufficient level of PK protein is required for cells to respond to AG-348 treatment ex-vivo, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients, ex vivo treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG-348 effectively upregulates PK enzymatic activity and increases stability in PK-deficient RBCs over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. ClinicalTrials.gov: NCT02476916, NCT03853798, NCT03548220, NCT03559699).
Assuntos
Eritrócitos , Piruvato Quinase , Trifosfato de Adenosina , Eritrócitos/metabolismo , Genótipo , Humanos , Piperazinas , Estabilidade Proteica , Piruvato Quinase/genética , QuinolinasRESUMO
Diagnosis of pyruvate kinase deficiency (PKD), the most common cause of hereditary non-spherocytic haemolytic anaemia, remains challenging in routine practice and no biomarkers for clinical severity have been characterised. This prospective study enrolled 41 patients with molecularly confirmed PKD from nine North American centres to evaluate the diagnostic sensitivity of pyruvate kinase (PK) enzyme activity and PK:hexokinase (HK) enzyme activity ratio, and evaluate the erythrocyte PK (PK-R) protein level and erythrocyte metabolites as biomarkers for clinical severity. In this population not transfused for ≥90 days before sampling, the diagnostic sensitivity of the PK enzyme assay was 90% [95% confidence interval (CI) 77-97%], whereas the PK:HK ratio sensitivity was 98% (95% CI 87-100%). There was no correlation between PK enzyme activity and clinical severity. Transfusion requirements correlated with normalised erythrocyte ATP levels (r = 0·527, P = 0·0016) and PK-R protein levels (r = -0·527, P = 0·0028). PK-R protein levels were significantly higher in the never transfused [median (range) 40·1 (9·8-73·9)%] versus ever transfused [median (range) 7·7 (0·4-15·1)%] patients (P = 0·0014). The PK:HK ratio had excellent sensitivity for PK diagnosis, superior to PKLR exon sequencing. Given that the number of PKLR variants and genotype combinations limits prognostication based on molecular findings, PK-R protein level may be a useful prognostic biomarker of disease severity and merits further study.
Assuntos
Anemia Hemolítica Congênita não Esferocítica/sangue , Eritrócitos/enzimologia , Hexoquinase/sangue , Piruvato Quinase/sangue , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/sangue , Adolescente , Adulto , Anemia Hemolítica Congênita não Esferocítica/genética , Biomarcadores/sangue , Criança , Pré-Escolar , Feminino , Hexoquinase/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/genética , Índice de Gravidade de DoençaRESUMO
Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Metabolismo Energético , Glucose/metabolismo , Ativação Linfocitária/imunologia , Metaboloma , Metabolômica , Animais , Proliferação de Células , Cromatografia Gasosa-Espectrometria de Massas , Glicólise , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Ativação Linfocitária/genética , Metabolômica/métodos , Camundongos , Estresse Oxidativo , Viroses/genética , Viroses/imunologia , Viroses/metabolismo , Viroses/virologiaRESUMO
Pyruvate kinase deficiency is a chronic hemolytic anemia caused by mutations in PK-R, a key glycolytic enzyme in erythrocytes. These 2 phase 1 randomized, placebo-controlled, double-blind healthy-volunteer studies assessed the safety, tolerability, and pharmacokinetics/pharmacodynamics of AG-348, a first-in-class allosteric PK-R activator. Twelve sequential cohorts were randomized 2:6 to receive oral placebo or AG-348, respectively, as a single dose (30-2500 mg) in the single-ascending-dose (SAD) study (ClinicalTrials.gov: NCT02108106) or 15-700 mg every 12 hours or 120 mg every 24 hours, for 14 days in the multiple-ascending-dose (MAD) study (ClinicalTrials.gov: NCT02149966). All 48 subjects completed the fasted SAD part; 44 of 48 completed the MAD (2 discontinued because of adverse events [AEs], 2 withdrew consent). The most common treatment-related AEs in AG-348-treated subjects were headache (16.7% [SAD] and 13.9% [MAD]) and nausea (13.9%, both studies). AE frequency increased at AG-348 doses ≥ 700 mg (SAD) and at 700 mg every 12 hours (MAD); 1 grade ≥ 3 AE occurred in the latter cohort. Pharmacokinetics were favorable with low variability. Dose-dependent changes in blood glycolytic intermediates consistent with glycolytic pathway activation were observed at all MAD doses, supporting future trials investigating the potential of AG-348 for treating PK deficiency or other anemias.
Assuntos
Piperazinas/administração & dosagem , Piperazinas/farmacocinética , Quinolinas/administração & dosagem , Quinolinas/farmacocinética , Adulto , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Feminino , Glicólise , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Piperazinas/efeitos adversos , Quinolinas/efeitos adversosRESUMO
Pyruvate kinase (PK) deficiency is a rare genetic disease that causes chronic hemolytic anemia. There are currently no targeted therapies for PK deficiency. Here, we describe the identification and characterization of AG-348, an allosteric activator of PK that is currently in clinical trials for the treatment of PK deficiency. We demonstrate that AG-348 can increase the activity of wild-type and mutant PK enzymes in biochemical assays and in patient red blood cells treated ex vivo. These data illustrate the potential for AG-348 to restore the glycolytic pathway activity in patients with PK deficiency and ultimately lead to clinical benefit.
Assuntos
Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/uso terapêutico , Eritrócitos/enzimologia , Piruvato Quinase/deficiência , Piruvato Quinase/metabolismo , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Anemia Hemolítica Congênita não Esferocítica , Animais , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/química , Eritrócitos/efeitos dos fármacos , Humanos , Cinética , Camundongos , Piperazinas , Piruvato Quinase/efeitos dos fármacos , Erros Inatos do Metabolismo dos Piruvatos , Quinolinas/química , Proteínas Recombinantes/metabolismo , Sulfonamidas/química , Doadores de TecidosRESUMO
The cytoprotective stress response factor HSF1 regulates the transcription of the chaperone HSP70, which exhibits anti-inflammatory effects and improves insulin sensitivity. We tested the therapeutic potential of this pathway in rodent models of diabetes using pharmacological tools. Activation of the HSF1 pathway was achieved using potent inhibitors of the upstream regulatory protein, HSP90. Treatment with AUY922, a selective HSP90 inhibitor led to robust inhibition of JNK1 phosphorylation, cytoprotection and improved insulin signaling in cells, consistent with effects observed with HSP70 treatment. Chronic dosing with HSP90 inhibitors reversed hyperglycemia in the diabetic db/db mouse model, and improved insulin sensitivity in the diet-induced obese mouse model of insulin resistance, further supporting the concept that the HSF1 pathway is a potentially viable anti-diabetes target.
Assuntos
Glicemia/efeitos dos fármacos , Proteínas de Ligação a DNA/agonistas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Hipoglicemiantes/administração & dosagem , Isoxazóis/administração & dosagem , Resorcinóis/administração & dosagem , Fatores de Transcrição/agonistas , Animais , Benzoquinonas/farmacologia , Glicemia/metabolismo , Células Cultivadas , Citoproteção , Diabetes Mellitus Tipo 2/metabolismo , Fatores de Transcrição de Choque Térmico , Resposta ao Choque Térmico , Isoxazóis/química , Lactamas Macrocíclicas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Resorcinóis/químicaRESUMO
BACKGROUND: Contrary to the traditional biology approach, where the expression patterns of a handful of genes are studied at a time, microarray experiments enable biologists to study the expression patterns of many genes simultaneously from gene expression profile data and decipher the underlying hidden biological mechanism from the observed gene expression changes. While the statistical significance of the gene expression data can be deduced by various methods, the biological interpretation of the data presents a challenge. RESULTS: A method, called CisTransMine, is proposed to help infer the underlying biological mechanisms for the observed gene expression changes in microarray experiments. Specifically, this method will predict potential cis-regulatory elements in promoter regions which could regulate gene expression changes. This approach builds on the MotifADE method published in 2004 and extends it with two modifications: up-regulated genes and down-regulated genes are tested separately and in addition, tests have been implemented to identify combinations of transcription factors that work synergistically. The method has been applied to a genome wide expression dataset intended to study myogenesis in a mouse C2C12 cell differentiation model. The results shown here both confirm the prior biological knowledge and facilitate the discovery of new biological insights. CONCLUSION: The results validate that the CisTransMine approach is a robust method to uncover the hidden transcriptional regulatory mechanisms that can facilitate the discovery of mechanisms of transcriptional regulation.
RESUMO
BACKGROUND: Using a gene clustering strategy we determined intracellular pathway relationships within skeletal myotubes in response to an acute heat stress stimuli. Following heat shock, the transcriptome was analyzed by microarray in a temporal fashion to characterize the dynamic relationship of signaling pathways. RESULTS: Bioinformatics analyses exposed coordination of functionally-related gene sets, depicting mechanism-based responses to heat shock. Protein turnover-related pathways were significantly affected including protein folding, pre-mRNA processing, mRNA splicing, proteolysis and proteasome-related pathways. Many responses were transient, tending to normalize within 24 hours. CONCLUSION: In summary, we show that the transcriptional response to acute cell stress is largely transient and proteosome-centric.
Assuntos
Regulação da Expressão Gênica , Transtornos de Estresse por Calor , Família Multigênica , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Camundongos , Fibras Musculares Esqueléticas/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma/análise , Transdução de Sinais/fisiologia , Transcrição GênicaRESUMO
The potential therapeutic value of resveratrol in age-related disease settings including cancer, diabetes, and Alzheimer's has emerged from a rapidly growing body of experimental evidence. Protection from oxidative stress appears to be a common feature of resveratrol that may be mediated through SirT1, though more specific molecular mechanisms by which resveratrol mediates its effects remain unclear. This has prompted an upsurge in cell-based mechanistic studies, often incorporating reporter assays for pathway elucidation in response to resveratrol treatment. Here, we report that resveratrol potently inhibits firefly luciferase with a K(i) value of 2microM, and caution that this confounding element may lead to compromised data interpretation.
Assuntos
Antioxidantes/farmacologia , Luciferases de Vaga-Lume/antagonistas & inibidores , Estilbenos/farmacologia , Animais , Linhagem Celular , Genes Reporter , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Camundongos , ResveratrolRESUMO
Stem cell differentiation is governed by extracellular signals that activate intracellular networks (or pathways) to drive phenotypic specification. Using a novel gene clustering strategy we determined pathway relationships from a genome-wide transcriptional dataset of skeletal myoblast differentiation. Established myogenic pathways, including cell contractility and cell-cycle arrest, were predicted with extreme statistical significance (p approximately 0). In addition, gene sets associated with angiogenesis, neuronal activity, and mRNA splicing were regulated, exposing developmental and therapeutic implications. Acquisition of transcriptional data spanning the entire differentiation time course provided context for a dynamic landscape of functional pathway regulation. This novel perspective on myogenic cell differentiation revealed previously unrecognized patterns of regulation. We predict that similar analyses will facilitate ongoing efforts to define molecular mechanisms in other stem cell and developmental paradigms. Finally, by combining an iterative process of analysis with supplementation of novel pathways, this application may evolve into a powerful discovery tool.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Desenvolvimento Muscular/genética , Mioblastos/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Animais , Ciclo Celular/genética , Perfilação da Expressão Gênica/métodos , Camundongos , Contração Muscular/fisiologia , Mioblastos/citologia , Neovascularização Fisiológica/genética , Neurônios/citologia , Neurônios/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células-Tronco/citologiaRESUMO
Much attention is focused on characterizing the contribution of bone marrow (BM)-derived cells to regenerating skeletal muscle, fuelled by hopes for stem cell-mediated therapy of muscle degenerative diseases. Though physical integration of BM stem cells has been well documented, little evidence of functional commitment to myotube phenotype has been reported. This is due to the innate difficulty in distinguishing gene products derived from donor versus host nuclei. Here, we demonstrate that BM-derived stem cells contribute via gene expression following incorporation to skeletal myotubes. By co-culturing human BM-derived mesenchymal stem cells (MSC) with mouse skeletal myoblasts, physical incorporation was observed by genetic lineage tracing and species-specific immunofluorescence. We used a human-specific antibody against the intermediate filament protein nestin, a marker of regenerating skeletal muscle, to identify functional contribution of MSC to myotube formation. Although nestin expression was never detected in MSC, human-specific expression was detected in myotubes that also contained MSC-derived nuclei. This induction of gene expression following myotube integration suggests that bone marrow-derived stem cells can reprogram and functionally contribute to the muscle cell phenotype. We propose that this model of myogenic commitment may provide the means to further characterize functional reprogramming of MSC to skeletal muscle.
Assuntos
Células da Medula Óssea/citologia , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Adipócitos/citologia , Animais , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular , Fusão Celular , Linhagem Celular , Linhagem da Célula , Núcleo Celular/metabolismo , Separação Celular/métodos , Técnicas de Cocultura , Endotélio Vascular/citologia , Imunofluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Mioblastos/citologia , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Nestina , Osteócitos/citologia , Especificidade da Espécie , Ativação TranscricionalRESUMO
CD154 (CD40L) mRNA turnover is regulated in part at the posttranscriptional level by a protein complex (termed Complex I) that binds to a highly CU-rich region of the 3'UTR. Polypyrimidine tract-binding protein (PTB) has previously been identified as a major RNA-binding protein in Complex I. Nondenaturing gel filtration of total extract from Jurkat T cells demonstrated that the CD154 mRNA-binding activity migrates as a approximately 200-kDa complex, indicating the presence of multiple complex-associated proteins. We have currently undertaken a biochemical approach to further characterize Complex I and observed that it segregates over DEAE-Sepharose into two subcomplexes (termed I-L and I-U). Furthermore, nucleolin was identified as a component of both subcomplexes and was shown that it is the major RNA-binding protein in I-U. To directly demonstrate the biological significance of Complex I binding to the CD154 transcript, cytoplasm from human Jurkat cells was fractionated over a sucrose gradient and the different cellular fractions subjected to immunoprecipitation with anti-PTB and anti-nucleolin Abs. RT-PCR of the immunoprecipitated products using CD154-specific primers clearly demonstrated that nucleolin and PTB are associated with CD154 mRNA in both the ribonucleoprotein and polysome fractions. These data strongly support a model whereby nucleolin and PTB are integral to the stability of CD154 mRNA and are components of the CD154 ribonucleoprotein particle associated with actively translating ribosomes.
Assuntos
Ligante de CD40/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linfócitos T/metabolismo , Ligante de CD40/biossíntese , Humanos , Substâncias Macromoleculares , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , NucleolinaRESUMO
Three HIF-alpha prolyl-4-hydroxylases (PHDs) (named PHD1, PHD2, and PHD3) effect the proteasome-mediated degradation of HIF by catalyzing the hydroxylation of key proline residues in the HIF-1 alpha subunit under normoxic conditions. When oxygen tension is reduced, PHD-mediated hydroxylation cannot occur, HIF-1 alpha accumulates in the nucleus, resulting in HIF-mediated gene transcription. In the present study, the expression and regulation of PHD mRNA and HIF protein expression was examined in human tissues and primary cells of cardiovascular origin. Treatment of human cardiac myocytes, smooth muscle cells, and endothelial cells with hypoxia or CoCl(2), a hypoxia mimic, resulted in a significant time-dependent increase in PHD3, but not PHD1 or PHD2, mRNA levels, which correlated with an increase in HIF-1 alpha protein expression. Overexpression studies revealed that PHD3 levels influence HIF-1 alpha stability in both normoxic and hypoxic conditions, suggesting that PHD3 may participate in a feedback loop controlling HIF activity.
Assuntos
Sistema Cardiovascular/metabolismo , Hipóxia/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Fatores de Transcrição/genética , Sequência de Bases , Hipóxia Celular/genética , Células Cultivadas , Cobalto/farmacologia , Primers do DNA/genética , Endotélio Vascular/metabolismo , Retroalimentação , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Cinética , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CD40 ligand (CD154) expression has been shown to be regulated, in part, at the posttranscriptional level by a pathway of "regulated instability" of mRNA decay throughout a time course of T cell activation. This pathway is modulated at late times of activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3' untranslated region of the CD154 message. We have undertaken experiments to extend these findings and to analyze the cis-acting elements and trans-acting factors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-rich motif. However, our current study shows that when this site was deleted additional complex binding was observed upstream and downstream of the minimal binding region. Only after deletion of an extended region (termed Delta1515) was complex binding completely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions bound related but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein. Deletion of the Delta1515 region resulted in reduced transcript stability as measured by both in vitro and in vivo decay assays. Finally, using Abs against known RNA-binding proteins, we identified the polypyrimidine tract-binding protein (or heterogeneous nuclear ribonucleoprotein I) as a candidate RNA-binding component of complex I.