RESUMO
Aqueously soluble oligomers of amyloid-ß peptide may be the principal neurotoxic forms of amyloid-ß in Alzheimer's disease, initiating downstream events that include tau hyperphosphorylation, neuritic/synaptic injury, microgliosis and neuron loss. Synthetic oligomeric amyloid-ß has been studied extensively, but little is known about the biochemistry of natural oligomeric amyloid-ß in human brain, even though it is more potent than simple synthetic peptides and comprises truncated and modified amyloid-ß monomers. We hypothesized that monoclonal antibodies specific to neurotoxic oligomeric amyloid-ß could be used to isolate it for further study. Here we report a unique human monoclonal antibody (B24) raised against synthetic oligomeric amyloid-ß that potently prevents Alzheimer's disease brain oligomeric amyloid-ß-induced impairment of hippocampal long-term potentiation. B24 binds natural and synthetic oligomeric amyloid-ß and a subset of amyloid plaques, but only in the presence of Ca2+. The amyloid-ß N terminus is required for B24 binding. Hydroxyapatite chromatography revealed that natural oligomeric amyloid-ß is highly avid for Ca2+. We took advantage of the reversible Ca2+-dependence of B24 binding to perform non-denaturing immunoaffinity isolation of oligomeric amyloid-ß from Alzheimer's disease brain-soluble extracts. Unexpectedly, the immunopurified material contained amyloid fibrils visualized by electron microscopy and amenable to further structural characterization. B24-purified human oligomeric amyloid-ß inhibited mouse hippocampal long-term potentiation. These findings identify a calcium-dependent method for purifying bioactive brain oligomeric amyloid-ß, at least some of which appears fibrillar.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos/metabolismo , Encéfalo/metabolismo , Cálcio/metabolismo , Humanos , Camundongos , Placa Amiloide/metabolismoRESUMO
Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of "developability." Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotype-matched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.
Assuntos
Anticorpos Monoclonais , Descoberta de Drogas/métodos , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Fenômenos Biofísicos , Aprovação de Drogas , Células HEK293 , Humanos , Imunoglobulina G/químicaRESUMO
Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs.
Assuntos
Anticorpos Monoclonais/farmacologia , Técnicas Biossensoriais/métodos , Receptor de Morte Celular Programada 1/imunologia , China , Desenvolvimento de Medicamentos , Epitopos/imunologia , União Europeia , Ensaios de Triagem em Larga Escala , Humanos , Receptor de Morte Celular Programada 1/química , Ligação Proteica , Ressonância de Plasmônio de Superfície , Estados UnidosRESUMO
Monoclonal antibodies (mAbs) have recently emerged as one of the most promising classes of biotherapeutics. A potential advantage of B cell-derived mAbs as therapeutic agents is that they have been subjected to natural filtering mechanisms, which may enrich for B cell receptors (BCRs) with favorable biophysical properties. Here, we evaluated 400 human mAbs for polyreactivity, hydrophobicity, and thermal stability using high-throughput screening assays. Overall, mAbs derived from memory B cells and long-lived plasma cells (LLPCs) display reduced levels of polyreactivity, hydrophobicity, and thermal stability compared with naive B cell-derived mAbs. Somatic hypermutation (SHM) is inversely associated with all three biophysical properties, as well as BCR expression levels. Finally, the developability profiles of the human B cell-derived mAbs are comparable with those observed for clinical mAbs, suggesting their high therapeutic potential. The results provide insight into the biophysical consequences of affinity maturation and have implications for therapeutic antibody engineering and development.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Humanos , Conformação MolecularRESUMO
Susceptibility of methionine to oxidation is an important concern for chemical stability during the development of a monoclonal antibody (mAb) therapeutic. To minimize downstream risks, leading candidates are usually screened under forced oxidation conditions to identify oxidation-labile molecules. Here we report results of forced oxidation on a large set of in-house expressed and purified mAbs with variable region sequences corresponding to 121 clinical stage mAbs. These mAb samples were treated with 0.1% H2O2 for 24 hours before enzymatic cleavage below the hinge, followed by reduction of inter-chain disulfide bonds for the detection of the light chain, Fab portion of heavy chain (Fd) and Fc by liquid chromatography-mass spectrometry. This high-throughput, middle-down approach allows detection of oxidation site(s) at the resolution of 3 distinct segments. The experimental oxidation data correlates well with theoretical predictions based on the solvent-accessible surface area of the methionine side-chains within these segments. These results validate the use of upstream computational modeling to predict mAb oxidation susceptibility at the sequence level.
Assuntos
Anticorpos Monoclonais/química , Peróxido de Hidrogênio/química , Espectrometria de Massas/métodos , Metionina/química , Modelos Moleculares , Cromatografia Líquida/métodos , Células HEK293 , Humanos , OxirreduçãoRESUMO
The state-of-the-art industrial drug discovery approach is the empirical interrogation of a library of drug candidates against a target molecule. The advantage of high-throughput kinetic measurements over equilibrium assessments is the ability to measure each of the kinetic components of binding affinity. Although high-throughput capabilities have improved with advances in instrument hardware, three bottlenecks in data processing remain: (1) intrinsic molecular properties that lead to poor biophysical quality in vitro are not accounted for in commercially available analysis models, (2) processing data through a user interface is time-consuming and not amenable to parallelized data collection, and (3) a commercial solution that includes historical kinetic data in the analysis of kinetic competition data does not exist. Herein, we describe a generally applicable method for the automated analysis, storage, and retrieval of kinetic binding data. This analysis can deconvolve poor quality data on-the-fly and store and organize historical data in a queryable format for use in future analyses. Such database-centric strategies afford greater insight into the molecular mechanisms of kinetic competition, allowing for the rapid identification of allosteric effectors and the presentation of kinetic competition data in absolute terms of percent bound to antigen on the biosensor.
Assuntos
Anticorpos/metabolismo , Automação Laboratorial/métodos , Processamento Eletrônico de Dados/métodos , Ensaios de Triagem em Larga Escala/métodos , Animais , Humanos , Cinética , Ligação ProteicaRESUMO
The differentiation of a preadipocyte into a mature adipocyte represents a fundamental process in biology that requires a scripted program of transcriptional events leading to changes in gene expression. As part of our contribution to the Nuclear Receptor Signaling Atlas (NURSA), we used quantitative real-time PCR to profile the temporal expression of all 49 members of the mouse nuclear receptor superfamily at selected time points during differentiation of 3T3-L1 cells into mature, lipid-bearing adipocytes using two differentiation inducers [DMI (a cocktail of dexamethasone, 3-isobutyl-1-methylxanthine, and insulin) and rosiglitazone]. We also included a comparative analysis of nuclear receptor expression in mouse primary preadipocytes and mature adipocytes. In addition to confirming the expression of receptors known to be required for adipogenesis, this analysis revealed the existence of a tightly regulated transcriptional cascade that appeared in three distinct temporal phases. The first phase began within 4 h of adipogenic initiation with the transient, sequential expression of four previously uncharacterized receptors, followed by biphasic expression of a second subset, and ended with the sequential increase in a third receptor subset over a period of 2 wk after initiation. The discovery that these receptors may serve as adipogenic biomarkers and as potential therapeutic targets in adipose-related diseases highlights the utility of quantitative expression profiling as a method for directing mechanism-based approaches to study complex regulatory pathways.
Assuntos
Adipócitos/citologia , Adipócitos/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Células 3T3-L1 , Animais , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Primers do DNA/genética , Bases de Dados de Proteínas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Real-time and label-free antibody screening systems are becoming more popular because of the increasing output of purified antibodies and antibody supernatant from many antibody discovery platforms. However, the properties of the biosensor can greatly affect the kinetic and epitope binning results generated by these label-free screening systems. ForteBio human-specific ProA, anti-human IgG quantitation (AHQ), anti-human Fc capture (AHC) sensors, and custom biotinylated-anti-human Fc capture (b-AHFc) sensors were evaluated in terms of loading ability, regeneration, kinetic characterization, and epitope binning with both purified IgG and IgG supernatant. AHC sensors proved unreliable for kinetic or binning assays at times, whereas AHQ sensors showed poor loading and regeneration abilities. ProA sensors worked well with both purified IgG and IgG supernatant. However, the interaction between ProA sensors and the Fab region of the IgG with VH3 germline limited the application of ProA sensors, especially in the epitope binning experiment. In an attempt to generate a biosensor type that would be compatible with a variety of germlines and sample types, we found that the custom b-AHFc sensors appeared to be robust working with both purified IgG and IgG supernatant, with little evidence of sensor-related artifacts.
Assuntos
Anticorpos Monoclonais/imunologia , Bioensaio/instrumentação , Bioensaio/métodos , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Epitopos/imunologia , Leveduras/imunologia , Biotinilação/métodos , Humanos , Imunoglobulina G/imunologia , CinéticaRESUMO
The importance of the neonatal Fc receptor (FcRn) in extending the serum half-life of monoclonal antibodies (mAbs) is well demonstrated, and has led to the development of multiple engineering approaches designed to alter Fc interactions with FcRn. Recent reports have additionally highlighted the effect of nonspecific interactions on antibody pharmacokinetics (PK), suggesting an FcRn-independent mechanism for mAb clearance. In this report we examine a case study of 2 anti-interleukin-12/23 antibodies, ustekinumab and briakinumab, which share the same target and Fc, but differ in variable region sequences. Ustekinumab displayed near baseline signal in a wide range of early stage developability assays for undesirable protein/protein interactions, while briakinumab showed significant propensity for self- and cross-interactions. This phenotypic difference correlates with faster clearance rates for briakinumab in both human FcRn transgenic and FcRn knockout mice. These findings support a dominant contribution for FcRn-independent clearance for antibodies with high nonspecificity, and highlight a key role for early stage developability screening to eliminate clones with such high nonspecific disposition PK.
Assuntos
Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Fc/imunologia , Ustekinumab/farmacocinética , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Ensaio de Imunoadsorção Enzimática , Humanos , Região Variável de Imunoglobulina/imunologia , Camundongos , Ustekinumab/imunologiaRESUMO
There is currently great interest in the fabrication of protein-detecting arrays comprised of large numbers of immobilized protein capture agents. While most efforts in this arena have focused on the use of biomolecules such as antibodies and nucleic acid aptamers as capture agents, synthetic species have many potential advantages. However, synthetic molecules isolated from combinatorial libraries generally do not bind target proteins with the high affinity necessary for array applications. Here, we demonstrate that simple linear peptides bind dimeric proteins tenaciously when immobilized, although they exhibit only modest affinity in solution. These data show that high-affinity bidentate capture agents for dimeric proteins can be created by simply immobilizing modest-affinity ligands on a surface at high density, bypassing the requirement for careful optimization of linker length and geometry that is normally required to create a high-affinity solution bidentate ligand.
Assuntos
Peptídeos/química , Análise Serial de Proteínas/métodos , Proteínas/química , Técnicas de Química Combinatória , Dimerização , Estabilidade de Medicamentos , Fluorescência , Ligantes , Biblioteca de PeptídeosRESUMO
Although improvements in technology for the isolation of potential therapeutic antibodies have made the process increasingly predictable, the development of biologically active monoclonal antibodies (mAbs) into drugs can often be impeded by developability issues such as poor expression, solubility, and promiscuous cross-reactivity. Establishing early stage developability screening assays capable of predicting late stage behavior is therefore of high value to minimize development risks. Toward this goal, we selected a panel of 16 monoclonal antibodies (mAbs) representing different developability profiles, in terms of self- and cross-interaction propensity, and examined their downstream behavior from expression titer to accelerated stability and pharmacokinetics in mice. Clearance rates showed significant rank-order correlations to 2 cross-interaction related assays, with the closest correlation to a non-specificity assay on the surface of yeast. Additionally, 2 self-association assays correlated with each other but not to mouse clearance rate. This case study suggests that combining assays capable of high throughput screening of self- and cross-interaction early in the discovery stage could significantly lower downstream development risks.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/farmacocinética , Imunoglobulina G/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Humanos , Imunoglobulina G/imunologia , Camundongos , Estabilidade ProteicaRESUMO
The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties.
Assuntos
Anticorpos Monoclonais/química , Ouro/química , Nanopartículas Metálicas/química , Cromatografia Líquida , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Solubilidade , Análise EspectralRESUMO
The discovery of monoclonal antibodies (mAbs) that bind to a particular molecular target is now regarded a routine exercise. However, the successful development of mAbs that (1) express well, (2) elicit a desirable biological effect upon binding, and (3) remain soluble and display low viscosity at high concentrations is often far more challenging. Therefore, high throughput screening assays that assess self-association and aggregation early in the selection process are likely to yield mAbs with superior biophysical properties. Here, we report an improved version of affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) that is capable of screening large panels of antibodies for their propensity to self-associate. AC-SINS is based on concentrating mAbs from dilute solutions around gold nanoparticles pre-coated with polyclonal capture (e.g., anti-Fc) antibodies. Interactions between immobilized mAbs lead to reduced inter-particle distances and increased plasmon wavelengths (wavelengths of maximum absorbance), which can be readily measured by optical means. This method is attractive because it is compatible with dilute and unpurified mAb solutions that are typical during early antibody discovery. In addition, we have improved multiple aspects of this assay for increased throughput and reproducibility. A data set comprising over 400 mAbs suggests that our modified assay yields self-interaction measurements that are well-correlated with other lower throughput assays such as cross-interaction chromatography. We expect that the simplicity and throughput of our improved AC-SINS method will lead to improved selection of mAbs with excellent biophysical properties during early antibody discovery.
Assuntos
Anticorpos Imobilizados/metabolismo , Anticorpos Monoclonais/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Imunoterapia/métodos , Análise Espectral/métodos , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Células Cultivadas , Descoberta de Drogas , Ouro/química , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/estatística & dados numéricos , Multimerização Proteica , Reprodutibilidade dos Testes , Ressonância de Plasmônio de SuperfícieRESUMO
Self-interaction of an antibody may lead to aggregation, low solubility or high viscosity. Rapid identification of highly developable leads remains challenging, even though progress has been made with the introduction of techniques such as self-interaction chromatography (SIC) and cross-interaction chromatography (CIC). Here, we report a high throughput method to detect antibody clone self-interaction (CSI) using bio-layer interferometry (BLI) technology. Antibodies with strong self-interaction responses in the CSI-BLI assay also show delayed retention times in SIC and CIC. This method allows hundreds of candidates to be screened in a matter of hours with minimal material consumption.
Assuntos
Anticorpos/metabolismo , Ensaios de Triagem em Larga Escala , InterferometriaRESUMO
Advances in human antibody discovery have allowed for the selection of hundreds of high affinity antibodies against many therapeutically relevant targets. This has necessitated the development of reproducible, high throughput analytical techniques to characterize the output from these selections. Among these characterizations, epitopic coverage and affinity are among the most critical properties for lead identification. Biolayer interferometry (BLI) is an attractive technique for epitope binning due to its speed and low antigen consumption. While surface-based methods such as BLI and surface plasmon resonance (SPR) are commonly used for affinity determinations, sensor chemistry and surface related artifacts can limit the accuracy of high affinity measurements. When comparing BLI and solution equilibrium based kinetic exclusion assays, significant differences in measured affinity (10-fold and above) were observed. KinExA direct association (k(a)) rate constant measurements suggest that this is mainly caused by inaccurate k(a) measurements associated with BLI related surface phenomena. Based on the kinetic exclusion assay principle used for KinExA, we developed a high throughput 96-well plate format assay, using a Meso Scale Discovery (MSD) instrument, to measure solution equilibrium affinity. This improved method combines the accuracy of solution-based methods with the throughput formerly only achievable with surface-based methods.
Assuntos
Afinidade de Anticorpos , Mapeamento de Epitopos/métodos , Ensaios de Triagem em Larga Escala/métodos , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Reações Antígeno-Anticorpo/imunologia , Antígenos/imunologia , Antígenos/metabolismo , Humanos , Cinética , Camundongos , Ligação Proteica , Padrões de Referência , SoluçõesRESUMO
The Bâ³/PR72 family of protein phosphatase 2A (PP2A) is an important PP2A family involved in diverse cellular processes, and uniquely regulated by calcium binding to the regulatory subunit. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here, we report crystal structures of the isolated PR72 and the trimeric PR70 holoenzyme at a resolution of 2.1 and 2.4 Å, respectively, and in vitro characterization of Cdc6 dephosphorylation. The holoenzyme structure reveals that one of the PR70 calcium-binding motifs directly contacts the scaffold subunit, resulting in the most compact scaffold subunit conformation among all PP2A holoenzymes. PR70 also binds distinctively to the catalytic subunit near the active site, which is required for PR70 to enhance phosphatase activity toward Cdc6. Our studies provide a structural basis for unique regulation of Bâ³/PR72 holoenzymes by calcium ions, and suggest the mechanisms for precise control of substrate specificity among PP2A holoenzymes.
Assuntos
Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Animais , Cristalografia por Raios X , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Fosforilação , Especificidade por SubstratoRESUMO
The molecular mechanism to regulate energy balance is not completely understood. Here we observed that Egr-1 expression in white adipose tissue (WAT) was highly correlated with dietary-induced obesity and insulin resistance both in mice and humans. Egr-1 null mice were protected from diet-induced obesity and obesity-associated pathologies such as fatty liver, insulin resistance, hyperlipidemia and hyperinsulinemia. This phenotype can be largely explained by the increase of energy expenditure in Egr-1 null mice. Characterization of these mice revealed that the expression of FOXC2 and its target genes were significantly elevated in white adipose tissues, leading to WAT energy expenditure instead of energy storage. Altogether, these studies suggest an important role for Egr-1, which, by repressing FOXC2 expression, promotes energy storage in WAT and favored the development of obesity under high energy intake.
Assuntos
Adipócitos/metabolismo , Dieta , Proteína 1 de Resposta de Crescimento Precoce/genética , Metabolismo Energético , Fatores de Transcrição Forkhead/genética , Obesidade/genética , Animais , Resistência à Insulina , Camundongos , Camundongos Knockout , Obesidade/etiologia , Obesidade/prevenção & controleRESUMO
Low expression, poor solubility, and polyspecificity are significant obstacles that have impeded the development of antibodies discovered from in vitro display libraries. Current biophysical characterization tools that identify these 'developability' problems are typically only applied after the discovery process, and thus limited to perhaps a few hundred candidates. We report a flow cytometric assay using a polyspecificity reagent (PSR) that allows for the identification and counter selection of polyspecific antibodies both during and after the selection process. The reported assay correlates well with cross-interaction chromatography, a surrogate for antibody solubility, as well as a baculovirus particle enzyme-linked immunosorbent assay, a surrogate for in vivo clearance. However, unlike these assays, PSR labeling is compatible both with screening of individual antibodies as well as selections of large antibody libraries. To this end, we demonstrate the ability to counter-select against polyspecificity while enriching for antigen affinity from a diverse antibody library, which enables simultaneous evolution of both antigen binding and superior non-target-related properties during the discovery process.
Assuntos
Anticorpos/genética , Técnicas de Visualização da Superfície Celular/métodos , Citometria de Fluxo , Leveduras/citologia , Anticorpos/química , Anticorpos/imunologia , Linhagem Celular , Membrana Celular/metabolismo , Reações Cruzadas , Evolução Molecular Direcionada , Humanos , Estabilidade Proteica , Solubilidade , TemperaturaRESUMO
Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of events including clonal expansion, growth arrest, and terminal differentiation. The mechanisms coordinating these different steps are not yet fully understood. Here we investigated whether microRNAs (miRNAs) play a role in this process. Microarray analysis was performed to detect miRNA expression during 3T3-L1 preadipocyte differentiation. Several miRNAs, including let-7, were up-regulated during 3T3-L1 adipogenesis. Ectopic introduction of let-7 into 3T3-L1 cells inhibited clonal expansion as well as terminal differentiation. The mRNA encoding high-mobility group AT-hook 2 (HMGA2), a transcription factor that regulates growth and proliferation in other contexts, was inversely correlated with let-7 levels during 3T3-L1 cell adipogenesis, and let-7 markedly reduced HMGA2 concentrations. Knockdown of HMGA2 inhibited 3T3-L1 differentiation. These results suggest that let-7 plays an important role in adipocyte differentiation and that it does so in part by targeting HMGA2, thereby regulating the transition from clonal expansion to terminal differentiation.
Assuntos
Adipogenia/genética , MicroRNAs/metabolismo , Células 3T3-L1 , Animais , Diferenciação Celular , Proliferação de Células , Células Clonais , Regulação da Expressão Gênica , Proteína HMGA2 , Camundongos , Modelos BiológicosRESUMO
Nuclear receptors (NRs) regulate gene expression in essential biological processes including differentiation and development. Here we report the systematic profiling of NRs in human and mouse embryonic stem cell (ESC) lines and during their early differentiation into embryoid bodies. Expression of the 48 human and mouse NRs was assessed by quantitative real-time PCR. In general, expression of NRs between the two human cell lines was highly concordant, whereas in contrast, expression of NRs between human and mouse ESCs differed significantly. In particular, a number of NRs that have been implicated previously as crucial regulators of mouse ESC biology, including ERRbeta, DAX-1, and LRH-1, exhibited diametric patterns of expression, suggesting they may have distinct species-specific functions. Taken together, these results highlight the complexity of the transcriptional hierarchy that exists between species and governs early development. These data should provide a unique resource for further exploration of the species-specific roles of NRs in ESC self-renewal and differentiation.