RESUMEN
It has been claimed that hyperestrogenism occurs in hypertrophic osteoarthropathy (HOA), but not in simple clubbing. However, one of our patients had simple clubbing and hyperestrogenism. We therefore measured estrogens, androgens, sex hormone-binding globulin (SHBG), and gonadotropins in five patients with HOA and in 18 patients with simple clubbing. Of the patients with HOA, 80% had a high urinary estriol concentration. In their serum, 80% had high estrone, 0% high estradiol, and 40% high SHBG. Of the patients with simple clubbing, 89% had a high urinary estriol concentration. In their serum, 76% had high estrone, 6% high estradiol, and 31% high SHBG. In all patients, urinary estriol concentration correlated positively with the degree of clubbing. Serum concentration of androstenedione, testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) was mostly normal, but androstenedione concentration correlated positively with the degree of clubbing. Spider angiomas were present in 74%, palmar erythema in 39%, and gynecomastia in 9%. Urinary creatinine concentration was low in 48% and correlated positively with the degree of clubbing. We reject the claim that hyperestrogenism occurs in HOA, but not in simple clubbing. Hyperestrogenism occurs both in HOA and in simple clubbing. Our results also support earlier reports that clubbing and HOA are associated with spider angiomas, palmar erythema, gynecomastia, adrenal cortical hyperfunction, muscle atrophy, and water retention. These results led to a new hypothesis on the pathogenesis of HOA, involving estrogens, prostaglandin E2, prostaglandin A2, and the inflammatory reflex.
Asunto(s)
Estrógenos/sangre , Dedos/patología , Osteoartropatía Hipertrófica Primaria/sangre , Osteoartropatía Hipertrófica Secundaria/sangre , Prostaglandinas/sangre , Adulto , Anciano , Creatinina/orina , Estriol/orina , Estrona/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Globulina de Unión a Hormona Sexual/análisisRESUMEN
BACKGROUND: Patients suffering from osteoporosis show an increased number of adipocytes in their bone marrow, concomitant with a reduction in the pool of human mesenchymal stem cells (hMSCs) that are able to differentiate into osteoblasts, thus leading to suppressed osteogenesis. METHODS: In order to be able to interfere with this process, we have investigated in-vitro culture conditions whereby adipogenic differentiation of hMSCs is impaired and osteogenic differentiation is promoted. By means of gene expression microarray analysis, we have investigated genes which are potential targets for prevention of fat cell differentiation. RESULTS: Our data show that BMP2 promotes both adipogenic and osteogenic differentiation of hMSCs, while transforming growth factor beta (TGFß) inhibits differentiation into both lineages. However, when cells are cultured under adipogenic differentiation conditions, which contain cAMP-enhancing agents such as IBMX of PGE2, TGFß promotes osteogenic differentiation, while at the same time inhibiting adipogenic differentiation. Gene expression and immunoblot analysis indicated that IBMX-induced suppression of HDAC5 levels plays an important role in the inhibitory effect of TGFß on osteogenic differentiation. By means of gene expression microarray analysis, we have investigated genes which are downregulated by TGFß under adipogenic differentiation conditions and may therefore be potential targets for prevention of fat cell differentiation. We thus identified nine genes for which FDA-approved drugs are available. Our results show that drugs directed against the nuclear hormone receptor PPARG, the metalloproteinase ADAMTS5, and the aldo-keto reductase AKR1B10 inhibit adipogenic differentiation in a dose-dependent manner, although in contrast to TGFß they do not appear to promote osteogenic differentiation. CONCLUSIONS: The approach chosen in this study has resulted in the identification of new targets for inhibition of fat cell differentiation, which may not only be relevant for prevention of osteoporosis, but also of obesity.
Asunto(s)
Adipogénesis/fisiología , Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Osteogénesis/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Adipocitos/metabolismo , Adipocitos/fisiología , Médula Ósea/metabolismo , Médula Ósea/fisiología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/fisiología , Linaje de la Célula/fisiología , Células Cultivadas , Dinoprostona/metabolismo , Expresión Génica/fisiología , Histona Desacetilasas/metabolismo , Humanos , Osteoblastos/metabolismo , Osteoblastos/fisiología , Osteoporosis/metabolismo , Osteoporosis/terapiaRESUMEN
BACKGROUND: MicroRNAs (miRNAs) are a family of small, non-coding single-stranded RNA molecules involved in post-transcriptional regulation of gene expression. As such, they are believed to play a role in regulating the step-wise changes in gene expression patterns that occur during cell fate specification of multipotent stem cells. Here, we have studied whether terminal differentiation of C2C12 myoblasts is indeed controlled by lineage-specific changes in miRNA expression. RESULTS: Using a previously generated RNA polymerase II (Pol-II) ChIP-on-chip dataset, we show differential Pol-II occupancy at the promoter regions of six miRNAs during C2C12 myogenic versus BMP2-induced osteogenic differentiation. Overexpression of one of these miRNAs, miR-378, enhances Alp activity, calcium deposition and mRNA expression of osteogenic marker genes in the presence of BMP2. CONCLUSIONS: Our results demonstrate a previously unknown role for miR-378 in promoting BMP2-induced osteogenic differentiation.
Asunto(s)
Proteína Morfogenética Ósea 2/genética , Diferenciación Celular/genética , Células Madre Mesenquimatosas/fisiología , MicroARNs/genética , Osteogénesis/genética , Animales , Línea Celular , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Mioblastos/metabolismo , Mioblastos/fisiología , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Network inference from gene expression data is a typical approach to reconstruct gene regulatory networks. During chondrogenic differentiation of human mesenchymal stem cells (hMSCs), a complex transcriptional network is active and regulates the temporal differentiation progress. As modulators of transcriptional regulation, microRNAs (miRNAs) play a critical role in stem cell differentiation. Integrated network inference aimes at determining interrelations between miRNAs and mRNAs on the basis of expression data as well as miRNA target predictions. We applied the NetGenerator tool in order to infer an integrated gene regulatory network. RESULTS: Time series experiments were performed to measure mRNA and miRNA abundances of TGF-beta1+BMP2 stimulated hMSCs. Network nodes were identified by analysing temporal expression changes, miRNA target gene predictions, time series correlation and literature knowledge. Network inference was performed using NetGenerator to reconstruct a dynamical regulatory model based on the measured data and prior knowledge. The resulting model is robust against noise and shows an optimal trade-off between fitting precision and inclusion of prior knowledge. It predicts the influence of miRNAs on the expression of chondrogenic marker genes and therefore proposes novel regulatory relations in differentiation control. By analysing the inferred network, we identified a previously unknown regulatory effect of miR-524-5p on the expression of the transcription factor SOX9 and the chondrogenic marker genes COL2A1, ACAN and COL10A1. CONCLUSIONS: Genome-wide exploration of miRNA-mRNA regulatory relationships is a reasonable approach to identify miRNAs which have so far not been associated with the investigated differentiation process. The NetGenerator tool is able to identify valid gene regulatory networks on the basis of miRNA and mRNA time series data.
Asunto(s)
Diferenciación Celular , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Condrocitos/citología , Condrogénesis , Biología Computacional , Regulación de la Expresión Génica , HumanosRESUMEN
This is the first study that comprehensively describes the effects of the platelet-derived growth factor (PDGF) isoforms C and D during in vitro expansion of human mesenchymal stem cells (hMSCs). Our results show that PDGFs can enhance proliferation of hMSCs without affecting their multipotency. It is of great value to culture and expand hMSCs in a safe and effective manner without losing their multipotency for manipulation and further development of cell-based therapies. Moreover, differential effects of PDGF isoforms have been observed on lineage-specific differentiation induced by BMP2 and Vitamin D3. Based on label-free LC-based quantitative proteomics approach we have furthermore identified specific pathways induced by PDGFs during the proliferation process, showing the importance of bioinformatics tools to study cell function.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Linfocinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteoma/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Linaje de la Célula , Colecalciferol/metabolismo , Humanos , Linfocinas/genética , Células Madre Mesenquimatosas/citología , Factor de Crecimiento Derivado de Plaquetas/genética , Proteoma/genética , Transcripción GenéticaRESUMEN
BACKGROUND: Inference of gene-regulatory networks (GRNs) is important for understanding behaviour and potential treatment of biological systems. Knowledge about GRNs gained from transcriptome analysis can be increased by multiple experiments and/or multiple stimuli. Since GRNs are complex and dynamical, appropriate methods and algorithms are needed for constructing models describing these dynamics. Algorithms based on heuristic approaches reduce the effort in parameter identification and computation time. RESULTS: The NetGenerator V2.0 algorithm, a heuristic for network inference, is proposed and described. It automatically generates a system of differential equations modelling structure and dynamics of the network based on time-resolved gene expression data. In contrast to a previous version, the inference considers multi-stimuli multi-experiment data and contains different methods for integrating prior knowledge. The resulting significant changes in the algorithmic procedures are explained in detail. NetGenerator is applied to relevant benchmark examples evaluating the inference for data from experiments with different stimuli. Also, the underlying GRN of chondrogenic differentiation, a real-world multi-stimulus problem, is inferred and analysed. CONCLUSIONS: NetGenerator is able to determine the structure and parameters of GRNs and their dynamics. The new features of the algorithm extend the range of possible experimental set-ups, results and biological interpretations. Based upon benchmarks, the algorithm provides good results in terms of specificity, sensitivity, efficiency and model fit.
Asunto(s)
Algoritmos , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/genética , Modelos Genéticos , Programas Informáticos , Simulación por Computador , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The ERBB1-ERBB4 receptors belong to a family of receptor tyrosine kinases that trigger a network of signaling pathways after ligand binding, thereby regulating cellular growth, differentiation and development. Ligand-induced signaling through ERBB1, also known as EGFR, is attenuated by the clathrin-dependent receptor-mediated endocytosis and RING E3-ligase Cbl-mediated receptor ubiquitination, which is followed by incorporation into multi-vesicular bodies (MVBs) and subsequent degradation in lysosomes. Before incorporation into MVBs, the EGFR is deubiquitinated by Usp8. We previously demonstrated that Usp8 is tyrosine phosphorylated in an EGFR- and SRC-kinase dependent manner. In the present study we show that overexpression of constitutively active SRC enhances constitutive and ligand-induced Usp8 tyrosine phosphorylation. We also show that enhanced endosomal recycling of the EGFR induced by TGFα stimulation is associated with decreased Usp8 tyrosine phosphorylation. We therefore hypothesize that tyrosine phosphorylation of Usp8 could regulate the function of Usp8. To identify Usp8 tyrosine phosphorylation site(s), we used Usp8 deletion constructs, site-directed mutagenesis of nine individual Usp8 tyrosine residues and mass spectrometry (MS) analysis. Our results demonstrate that the MIT-domain is necessary for ligand-induced tyrosine phosphorylation of Usp8 1-504. However, mutation of three MIT domain tyrosine residues did not abolish Usp8 tyrosine phosphorylation. Similar results were obtained upon mutation of six exposed tyrosine residues in the Rhod domain and linker region. Repeated MS analysis of both Usp8 WT and C748A mutants readily detected serine phosphorylation, including the S680 14-3-3 binding site, but did not reveal any phospho-tyrosine residues. Notably, mutation of the tyrosine residue in the Usp8 14-3-3 binding motif (Y679) did not abolish phosphoserine-dependent binding of 14-3-3 to Usp8. Our findings are most consistent with the model that MIT domain-dependent recruitment of Usp8 to endosomal membranes is important for low stoichiometry SRC-mediated tyrosine phosphorylation of multiple Usp8 tyrosines. Our findings demonstrate that Usp8 is a target for the post-translational serine and tyrosine phosphorylation, most likely characterized by low abundant tyrosine phosphorylation on multiple residues, and high abundant serine phosphorylation on several residues.
Asunto(s)
Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Serina/metabolismo , Tirosina/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Proteínas 14-3-3/metabolismo , Endopeptidasas/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Mutagénesis , Fosforilación/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , Factor de Crecimiento Transformador alfa/metabolismo , Ubiquitina Tiolesterasa/química , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
ERBB receptors have an important function in mammalian development and normal physiology, but overexpression and poor downregulation of ERBB receptors have been associated with malignant growth. Ligand-induced ERBB receptor signaling is terminated by clathrin-dependent receptor endocytosis, followed by incorporation of activated receptor complexes into multi-vesicular bodies and subsequent degradation in lysosomes. In the case of ERBB1, also known as the EGF receptor, it has been shown that ubiquitination serves as a signal to facilitate internalization and subsequent endosomal sorting, but little is known about the role of ubiquitination of other ERBB receptors. In the present study we investigated the regulation of ubiquitination and deubiquitination of the ERBB4 CYT-1 and CYT-2 isoforms in the context of chimeric EGFR-ERBB4 receptors. We demonstrate that EGFR-ERBB4 CYT-2 chimera shows decreased ligand-induced downregulation and EGF-degradation, as well as enhanced EGF recycling, when compared to EGFR-ERBB4 CYT-1. Moreover we show that the mutation Y1103F in the binding site for Cbl which is present in both CYT-1 and CYT-2, does not influence ERBB4 endosomal trafficking. We further demonstrate that total ligand-induced ubiquitination of CYT-1 is higher than that of CYT-2, whereby CYT-1 ubiquitination is mainly dependent on the PPXY(1056) Itch binding site for the E3-ligase Itch which is only present in CYT-1, while that of CYT-2 is dependent on the Y1103 Cbl binding site. The E3-ligase c-Cbl is more efficiently phosphorylated upon EGF stimulation of the CYT-2 than the CYT-1 isoform. Moreover our data show that the pY1103 Cbl binding site is required for K48-polyubiquitination of both CYT-1 and CYT-2, whereas the PPXY(1056) Itch binding site is required for K63-polyubiquitination of CYT-1. We further demonstrate that EGF stimulation of EGFR-ERBB4 CYT-1 and CYT-2 does not result in efficient binding to and tyrosine phosphorylation of the ESCRT-0 subunit Hrs. Finally, even though CYT-1 shows ligand-induced K63-polyubiquitination, it is not subjected to deubiquitination by the K63 polyubiquitin-specific AMSH deubiquitinating enzyme, while CYT-1 is slightly deubiquitinated by USP8. We conclude that Cbl and Itch binding sites in ERBB4 CYT-1 and CYT-2 mediate K48- and K63-polyubiquitination, respectively.
Asunto(s)
Receptores ErbB/metabolismo , Lisina/química , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Sitios de Unión , Línea Celular , Regulación hacia Abajo , Endosomas/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor ErbB-4 , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Transfección , Ubiquitinación/efectos de los fármacosRESUMEN
DNA methylation is an important epigenetic regulator of gene expression. Abnormalities in DNA methylation patterns have been associated with various developmental and proliferative diseases, particularly cancer. Targeting DNA methyltransferases (DNMTs) represents a promising strategy for the treatment of such diseases. Current DNMT inhibitors suffer important drawbacks with respect to their efficacy, specificity, and toxicity. In this study, we have set up a robust in vitro bacterial M.SssI DNMT activity assay to systematically screen a collection of 26 240 compounds that were predicted to compete with the S-adenosyl-L-methionine (SAM) substrate of DNMT. This resulted in the identification of a novel set of structurally distinct inhibitors of M.SssI DNMT activity. Although molecular docking studies using an M.SssI homology model suggest that these compounds might compete with SAM binding, mode of activity (MoA) assays are still needed to confirm this hypothesis. Our set of novel M.SssI DNMT inhibitors, once confirmed in an orthogonal DNMT assay, may thus serve as a starting point to identify and characterize suitable lead candidates for further drug optimization.
Asunto(s)
Metilación de ADN/efectos de los fármacos , ADN-Citosina Metilasas/antagonistas & inhibidores , ADN-Citosina Metilasas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/química , ADN Bacteriano/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
ErbB receptors play an important role in normal cellular growth, differentiation and development, but overexpression or poor downregulation can result in enhanced signaling and cancerous growth. ErbB signaling is terminated by clathrin-dependent receptor-mediated endocytosis, followed by incorporation in multi-vesicular bodies and subsequent degradation in lysosomes. In contrast to EGFR, ErbB2 displays poor ligand-induced downregulation and enhanced recycling, but the molecular mechanisms underlying this difference are poorly understood. Given our previous observation that both EGFR and an EGFR-ErbB2 chimera undergo Cbl-mediated K63-polyubiquitination, we investigated in the present study whether activation of the EGFR and the EGFR-ErbB2 chimera is associated with tyrosine phosphorylation of the ESCRT-0 complex subunit Hrs and AMSH-mediated deubiquitination. EGF stimulation of the EGFR resulted in efficient Hrs tyrosine phosphorylation and deubiquitination by the K63-polyubiquitin chain-specific deubiquitinating enzyme AMSH. In contrast, EGF activation of EGFR-ErbB2 showed significantly decreased Hrs tyrosine phosphorylation and deubiquitination by AMSH. To test whether this phenotype is the result of endosomal recycling, we induced recycling of the EGFR by stimulation with TGFα. Indeed, even though TGFα-stimulation of EGFR is associated with efficient ligand-stimulated K63-polyubiquitination, we observed that Hrs tyrosine phosphorylation as well as AMSH-mediated deubiquitination is significantly reduced under these conditions. Using various EGFR-ErbB2 chimeras, we demonstrate that enhanced recycling, decreased Hrs tyrosine phosphorylation and decreased AMSH mediated deubiquitination of EGFR-ErbB2 chimeras is primarily due to the presence of ErbB2 sequences or the absence of EGFR sequences C-terminal to the Cbl binding site. We conclude that endosomal recycling of the EGFR and ErbB2 receptors is associated with significantly impaired tyrosine phosphorylation of the ESCRT-0 subunit Hrs as well as decreased deubiquitination by AMSH, which is consistent with the finding that recycling receptors are not efficiently incorporated in the MVB pathway.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Receptores ErbB/metabolismo , Fosfoproteínas/metabolismo , Receptor ErbB-2/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Regulación hacia Abajo/efectos de los fármacos , Endocitosis , Endosomas/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transfección , Factor de Crecimiento Transformador alfa/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation-contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 µM of the DNA methylation inhibitor 5-azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up-regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC-treated myotubes displayed a mature, well-defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 µM ryanodine, and 100 µM nifedipine, suggesting that action potential-induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.
Asunto(s)
Azacitidina/farmacología , Metilación de ADN/fisiología , Desarrollo de Músculos/efectos de los fármacos , Músculo Esquelético/citología , Animales , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Quelantes/farmacología , Metilación de ADN/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Epigénesis Genética , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas , Ratones , Contracción Muscular/efectos de los fármacos , Fibras Musculares Esqueléticas , Nifedipino/farmacología , Rianodina/farmacología , Sarcómeros/efectos de los fármacos , Sarcómeros/ultraestructura , Tetrodotoxina/farmacologíaRESUMEN
The progressive restriction of differentiation potential from pluripotent embryonic stem cells, via multipotent progenitor cells to terminally differentiated, mature somatic cells, involves step-wise changes in transcription patterns that are tightly controlled by the coordinated action of key transcription factors and changes in epigenetic modifications. While previous studies have demonstrated tissue-specific differences in DNA methylation patterns that might function in lineage restriction, it is unclear at what exact developmental stage these differences arise. Here, we have studied whether terminal, multi-lineage differentiation of C2C12 myoblasts is accompanied by lineage-specific changes in DNA methylation patterns. Using bisulfite sequencing and genome-wide methylated DNA- and chromatin immunoprecipitation-on-chip techniques we show that in these cells, in general, myogenic genes are enriched for RNA polymerase II and hypomethylated, whereas osteogenic genes show lower polymerase occupancy and are hypermethylated. Removal of DNA methylation marks by 5-azacytidine (5AC) treatment alters the myogenic lineage commitment of these cells and induces spontaneous osteogenic and adipogenic differentiation. This is accompanied by upregulation of key lineage-specific transcription factors. We subsequently analyzed genome-wide changes in DNA methylation and polymerase II occupancy during BMP2-induced osteogenesis. Our data indicate that BMP2 is able to induce the transcriptional program underlying osteogenesis without changing the methylation status of the genome. We conclude that DNA methylation primes C2C12 cells for myogenesis and prevents spontaneous osteogenesis, but still permits induction of the osteogenic transcriptional program upon BMP2 stimulation. Based on these results, we propose that cell type-specific DNA methylation patterns are established prior to terminal differentiation of adult progenitor cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Animales , Azacitidina/farmacología , Diferenciación Celular/genética , Linaje de la Célula/genética , Inmunoprecipitación de Cromatina , Metilación de ADN/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Desarrollo de Músculos/efectos de los fármacos , Desarrollo de Músculos/genética , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Factor de Transcripción Sp7 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
In myasthenia gravis (MG), the neuromuscular transmission is impaired mainly by auto-antibodies against the acetylcholine receptor (AChR) or MuSK. In about 5% of the MG patients, however, the auto-antigen is still unknown. We investigated whether these idiopathic MG patients (iMG) have auto-antibodies against ErbB proteins, which influence the AChR density at the NMJ. Our results show reduced mRNA expression levels of ErbB4 in thymus tissue of iMG patients compared to AChR-MG and non-MG patients, but we could not detect anti-ErbB antibodies in sera of iMG patients. Therefore, our results do not support a role for ErbB receptors as auto-antigens in iMG patients.
Asunto(s)
Autoanticuerpos/sangre , Receptores ErbB/biosíntesis , Receptores ErbB/inmunología , Miastenia Gravis/inmunología , Timo/inmunología , Adolescente , Adulto , Anciano , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoantígenos/metabolismo , Separación Celular , Niño , Femenino , Citometría de Flujo , Humanos , Immunoblotting , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Miastenia Gravis/metabolismo , ARN Mensajero/análisis , Sinapsis/inmunología , Timo/metabolismo , Adulto JovenRESUMEN
Bone marrow-derived human mesenchymal stem cells (hMSCs) have the in vitro capacity to differentiate into osteoblasts, chondrocytes or adipocytes, depending on the applied stimulus. In order to identify novel regulators of osteogenesis in hMSCs, osteo-transcriptomics was performed whereby differentiation induced by dexamethasone (DEX), DEX+ bone morphogenetic protein 2 (BMP2), and DEX+ Vitamin D(3) (1,25(OH)(2)D(3)) was studied over a course of 12 days. Microarray analysis revealed that 2095 genes were significantly regulated by DEX+ 1,25(OH)(2)D(3), of which 961 showed accelerated expression kinetics compared to treatment by DEX alone. The majority of these genes were accelerated 24-48 h after onset of osteogenic treatment. Gene ontology (GO) analysis of these 1,25(OH)(2)D(3)-accelerated genes indicated their involvement in biological processes related to cellular differentiation and cell cycle regulation. When compared to cells treated with DEX or DEX+BMP2, treatment with DEX+ 1,25(OH)(2)D(3) clearly accelerated osteoprogenitor commitment and osteoblast maturation, as measured by alkaline phosphatase (ALP) activity and calcification of the matrix. Cell cycle progression, as observed after initial growth arrest, was not significantly accelerated by 1,25(OH)(2)D(3) and was not required for onset and progression of osteogenesis. However, expression of c-Myc was accelerated by 1,25(OH)(2)D(3), and binding sites for c-MYC were enriched in promoters of genes accelerated by 1,25(OH)(2)D(3). Lentiviral overexpression of c-MYC strongly promoted DEX+ BMP2-induced osteoblast differentiation and matrix maturation. In conclusion, our studies show for the first time that 1,25(OH)(2)D(3) strongly accelerates expression of genes involved in differentiation of hMSCs and, moreover, identify c-MYC as a novel regulator of osteogenesis.
Asunto(s)
Proteína Morfogenética Ósea 2/fisiología , Diferenciación Celular/genética , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Osteogénesis/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , Vitamina D/fisiología , Proteína Morfogenética Ósea 2/genética , Calcificación Fisiológica/genética , Células Cultivadas , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Osteoblastos/citología , Osteoblastos/fisiología , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Regulación hacia Arriba/genéticaRESUMEN
The epidermal growth factor (EGF)-like growth factors bind their ErbB receptors in a highly selective manner. Recently, we have shown that the sequence YYDLL in the C-terminal linear region is compatible with binding to all ligand-binding ErbB receptors. In the present study, we show that introduction of the YYDLL sequence into the ErbB1 specific ligands EGF and transforming growth factor-alpha (TGFalpha) broadened their receptor specificity towards ErbB4. Upon introduction of the YYDLL sequence into epiregulin, which by itself binds ErbB1 and ErbB4 but not ErbB3, its binding specificity was broadened to ErbB3, concomitant with enhanced affinity for ErbB4. Introduction of the YYDLL sequence into NRG1beta resulted in a 10-fold increase in affinity for ErbB3, without affecting its receptor specificity. Remarkably, the strongly enhanced affinity for ErbB3 negatively influenced their mitogenic activity towards cells coexpressing ErbB2 and ErbB3. These observations are discussed in terms of the optimised ErbB affinity, selectivity and mitogenic potential that have taken place during evolution.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Neurregulina-1/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Epirregulina , Receptores ErbB/metabolismo , Genes erbB-1/fisiología , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Receptor ErbB-2/metabolismo , Receptor ErbB-4RESUMEN
Poor downregulation of ErbB receptors is associated with enhanced downstream signaling and tumorigenesis. It has been suggested that poor downregulation of ErbB-2, -3 and -4 receptors when compared to ErbB1 is due to decreased recruitment of Cbl E3 ligase proteins. However, a highly conserved Cbl binding site is not only present in ErbB1/EGFR (FLQRpY(1045)SSDP), but also in ErbB2 (PLQRpY(1091)SEDP) and ErbB4 (STQRpY(1103)SADP). We therefore replaced the ErbB1 Cbl binding site by that of ErbB2 and ErbB4. Whereas retrovirally infected NIH3T3 cells containing the EGFR Y1045F mutation showed dramatically impaired Cbl recruitment, EGFR ubiquitination and delayed EGFR degradation, replacement of the EGFR Cbl binding site by that of ErbB2 or ErbB4 did not affect Cbl recruitment, receptor-ubiquitination, -degradation, -downregulation or ligand degradation. We conclude that poor downregulation of ErbB2 and ErbB4 receptors is not due to sequence variations in the Cbl binding site of these receptors.
Asunto(s)
Receptores ErbB/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Receptor ErbB-2/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Regulación hacia Abajo , Endosomas/enzimología , Humanos , Ratones , Células 3T3 NIH , Receptor ErbB-4 , Transducción de Señal , UbiquitinaciónRESUMEN
BACKGROUND: Neural tube defects are multifactorial malformations involving both environmental exposures, such as maternal nutrition, and genetic factors. Aberrant expression of the platelet-derived growth factor alpha-receptor (PDGFRA) gene has been implicated in neural-tube-defect etiology in both mice and humans. METHODS: We investigated possible interactions between the PDGFRA promoter haplotype of mother and child, as well as maternal glucose, myo-inositol, and zinc levels, in relation to spina bifida offspring. Distributions were determined of the PDGFRA promoter haplotypes H1 and H2 in a Dutch cohort, consisting of 88 spina bifida children with 56 of their mothers, and 74 control children with 72 of their mothers, as well as maternal plasma glucose, myo-inositol, and red blood cell zinc concentrations. RESULTS: A significantly higher frequency of H1 was observed in children with spina bifida than in controls (30.1 vs. 20.3%; OR = 1.69, 95% CI 1.02-2.83). High maternal body mass index (BMI) and glucose were significant risk factors for both H1 and H2 children, whereas low myo-inositol and zinc were risk factors for H2 but not for H1 children. Stepwise multiple logistic regression analysis showed that high maternal glucose and low myo-inositol are the main risk factors for H2 spina bifida children, whereas for H1 spina bifida children, maternal BMI was the main risk factor. Interestingly, H1 mothers (median 165.5 cm) showed a significantly lower body height than H2 mothers (median 169.1 cm; p = 0.003). CONCLUSIONS: These data suggest that the child's PDGFRA promoter haplotype is differentially sensitive for periconceptional exposure to glucose, myo-inositol, and zinc in the risk of spina bifida.
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Exposición a Riesgos Ambientales , Exposición Materna , Regiones Promotoras Genéticas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Disrafia Espinal/genética , Estudios de Casos y Controles , Niño , Estudios de Cohortes , Femenino , Haplotipos , Humanos , Modelos Logísticos , Riesgo , Disrafia Espinal/epidemiologíaRESUMEN
ErbB3 transactivation can make tumor cells resistance to ErbB1/ErbB2 targeting drugs. This urges for a reliable method to determine cell surface ErbB3 levels, but in our hands iodinated NRG1beta is unstable and tends to underestimate the number of ErbB3 receptors in a radio-receptor assay. Here we show by the use of a radio-labeled high affinity neuregulin mutant NRG/YYDLL that ErbB3 levels can be determined in a reliable manner by Scatchard analysis. Furthermore we show by differential competition with unlabeled NRG/YYDLL and betacellulin that the number of ErbB3 and ErbB4 receptors can be quantified separately on cultured human breast cancer cells.
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Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos , Proteínas del Tejido Nervioso/metabolismo , Receptor ErbB-3/análisis , Animales , Betacelulina , Unión Competitiva , Western Blotting , Receptores ErbB/análisis , Receptores ErbB/metabolismo , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Radioisótopos de Yodo/química , Marcaje Isotópico , Ratones , Mutación , Proteínas del Tejido Nervioso/genética , Neurregulina-1 , Reacción en Cadena de la Polimerasa , Ensayo de Unión Radioligante , Receptor ErbB-3/metabolismo , Receptor ErbB-4 , Células Tumorales CultivadasRESUMEN
Aberrant expression of the platelet-derived growth factor alpha-receptor (PDGFRA) gene has been associated with various diseases, including neural tube defects and gliomas. We have previously identified 5 distinct haplotypes for the PDGFRA promoter region, designated H1, H2alpha, H2beta, H2gamma and H2delta. Of these haplotypes H1 and H2alpha are the most common, whereby H1 drives low and H2alpha high transcriptional activity in transient transfection assays. Here we have investigated the role of these PDGFRA promoter haplotypes in gliomagenesis at both the genetic and cellular level. In a case-control study on 71 glioblastoma patients, we observed a clear underrepresentation of H1 alleles, with pH1 = 0.141 in patients and pH1 = 0.211 in a combined Western European control group (n = 998, p < 0.05). Furthermore, in 3 out of 4 available H1/H2alpha heterozygous human glioblastoma cell lines, H1-derived mRNA levels were more than 10-fold lower than from H2alpha, resulting at least in part from haplotype-specific epigenetic differences such as DNA methylation and histone acetylation. Together, these results indicate that PDGFRA promoter haplotypes may predispose to gliomas. We propose a model in which PDGFRA is upregulated in a haplotype-specific manner during neural stem cell differentiation, which affects the pool size of cells that can later undergo gliomagenesis.
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Glioblastoma/química , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Acetilación , Adulto , Anciano , Estudios de Casos y Controles , Metilación de ADN , Electroforesis en Gel de Poliacrilamida , Europa (Continente)/epidemiología , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Glioblastoma/epidemiología , Glioblastoma/genética , Haplotipos , Histonas/metabolismo , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Medición de Riesgo , Factores de Riesgo , Regulación hacia ArribaRESUMEN
Density-dependent growth inhibition secures tissue homeostasis. Dysfunction of the mechanisms, which regulate this type of growth control is a major cause of neoplasia. In confluent normal rat kidney (NRK) fibroblasts, epidermal growth factor (EGF) receptor levels decline, ultimately rendering these cells irresponsive to EGF. Using an activator protein (AP)-1 sensitive reporter construct, we show that AP-1 activity is strongly decreased in density-arrested NRK cells, but is restored after relaxation of densitydependent growth inhibition by removing neighboring cells. EGF could not induce AP-1 activity or S-phase entry in density-arrested cells, but could do so after pretreatment with retinoic acid, which enhances EGF receptor expression. Our results support a model in which the EGF receptor regulates density-dependent growth control in NRK fibroblasts, which is reflected by EGF-induced mitogenic signaling and consequent AP-1 activity.