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1.
Stem Cells ; 36(1): 55-64, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29047185

RESUMEN

Hematopoietic stem cells derived from pluripotent stem cells could be used as an alternative to bone marrow transplants. Deriving these has been a long-term goal for researchers. However, the success of these efforts has been limited with the cells produced able to engraft in the bone marrow of recipient animals only in very low numbers. There is evidence that defects in the migratory and homing capacity of the cells are due to mis-regulation of miRNA expression and are responsible for their failure to engraft. We compared the miRNA expression profile of hematopoietic progenitors derived from pluripotent stem cells to those derived from bone marrow and found that numerous miRNAs are too highly expressed in hematopoietic progenitors derived from pluripotent stem cells, and that most of these are inhibitors of epithelial-mesenchymal transition or metastasis (including miR-200b, miR-200c, miR-205, miR-148a, and miR-424). We hypothesize that the high expression of these factors, which promote an adherent phenotype, may be causing the defect in hematopoietic differentiation. However, inhibiting these miRNAs, individually or in multiplex, was insufficient to improve hematopoietic differentiation in vitro, suggesting that other miRNAs and/or genes may be involved in this process. Stem Cells 2018;36:55-64.


Asunto(s)
Transición Epitelial-Mesenquimal/genética , Células Madre Hematopoyéticas/metabolismo , MicroARNs/genética , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Regulación hacia Abajo , Humanos
2.
Stem Cells ; 31(5): 1022-9, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23280624

RESUMEN

Fanconi anemia (FA) is a genomic instability disorder caused by mutations in genes involved in replication-dependant-repair and removal of DNA cross-links. Mouse models with targeted deletions of FA genes have been developed; however, none of these exhibit the human bone marrow aplasia. Human embryonic stem cell (hESC) differentiation recapitulates many steps of embryonic hematopoietic development and is a useful model system to investigate the early events of hematopoietic progenitor specification. It is now possible to derive patient-specific human-induced pluripotent stem cells (hiPSC); however, this approach has been rather difficult to achieve in FA cells due to a requirement for activation of FA pathway during reprogramming process which can be bypassed either by genetic complementation or reprogramming under hypoxic conditions. In this study, we report that FA-C patient-specific hiPSC lines can be derived under normoxic conditions, albeit at much reduced efficiency. These disease-specific hiPSC lines and hESC with stable knockdown of FANCC display all the in vitro hallmarks of pluripotency. Nevertheless, the disease-specific hiPSCs show a much higher frequency of chromosomal abnormalities compared to parent fibroblasts and are unable to generate teratoma composed of all three germ layers in vivo, likely due to increased genomic instability. Both FANCC-deficient hESC and hiPSC lines are capable of undergoing hematopoietic differentiation, but the hematopoietic progenitors display an increased apoptosis in culture and reduced clonogenic potential. Together these data highlight the critical requirement for FA proteins in survival of hematopoietic progenitors, cellular reprogramming, and maintenance of genomic stability.


Asunto(s)
Reprogramación Celular/fisiología , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Células Madre Hematopoyéticas/patología , Células Madre Pluripotentes Inducidas/patología , Diferenciación Celular/fisiología , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Terapia Genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo
3.
Stem Cells ; 31(9): 2015-23, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23818183

RESUMEN

Cernunnos (also known as XLF) deficiency syndrome is a rare recessive autosomal disorder caused by mutations in the XLF gene, a key factor involved in the end joining step of DNA during nonhomologous end joining (NHEJ) process. Human patients with XLF mutations display microcephaly, developmental and growth delays, and severe immunodeficiency. While the clinical phenotype of DNA damage disorders, including XLF Syndrome, has been described extensively, the underlying mechanisms of disease onset, are as yet, undefined. We have been able to generate an induced pluripotent stem cell (iPSC) model of XLF deficiency, which accurately replicates the double-strand break repair deficiency observed in XLF patients. XLF patient-specific iPSCs (XLF-iPSC) show typical expression of pluripotency markers, but have altered in vitro differentiation capacity and an inability to generate teratomas comprised of all three germ layers in vivo. Our results demonstrate that XLF-iPSCs possess a weak NHEJ-mediated DNA repair capacity that is incapable of coping with the DNA lesions introduced by physiological stress, normal metabolism, and ionizing radiation. XLF-iPSC lines are capable of hematopoietic differentiation; however, the more primitive subsets of hematopoietic progenitors display increased apoptosis in culture and an inability to repair DNA damage. Together, our findings highlight the importance of NHEJ-mediated-DNA repair in the maintenance of a pristine pool of hematopoietic progenitors during human embryonic development.


Asunto(s)
Enzimas Reparadoras del ADN/deficiencia , Proteínas de Unión al ADN/deficiencia , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Secuencia de Bases , Diferenciación Celular , Línea Celular , Supervivencia Celular , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Datos de Secuencia Molecular
4.
Stem Cells ; 30(4): 599-611, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22311747

RESUMEN

Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species. In this study, we reprogrammed three normal unrelated patient's fibroblasts (p47(phox) and gp91(phox) ) to pluripotency by lentiviral transduction with defined pluripotency factors. These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors, and possess high telomerase activity. Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo. They were isogenic with the original patient fibroblasts, exhibited normal karyotype, and retained the p47(phox) or gp91(pho) (x) mutations found in the patient fibroblasts. We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions. Most importantly, CGD-patient-specific iPSC-derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients. Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modeling CGD disease phenotypes, screening candidate drugs, and the development of gene therapy.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Enfermedad Granulomatosa Crónica/patología , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Diferenciación Celular , Línea Celular , Citocinas/metabolismo , Humanos , Cariotipificación , Cinética , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/metabolismo , NADPH Oxidasas/metabolismo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo
5.
Sci Adv ; 8(12): eabk0013, 2022 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-35333572

RESUMEN

Uncovering the mechanisms that establish naïve pluripotency in humans is crucial for the future applications of pluripotent stem cells including the production of human blastoids. However, the regulatory pathways that control the establishment of naïve pluripotency by reprogramming are largely unknown. Here, we use genome-wide screening to identify essential regulators as well as major impediments of human primed to naïve pluripotent stem cell reprogramming. We discover that factors essential for cell state change do not typically undergo changes at the level of gene expression but rather are repurposed with new functions. Mechanistically, we establish that the variant Polycomb complex PRC1.3 and PRDM14 jointly repress developmental and gene regulatory factors to ensure naïve cell reprogramming. In addition, small-molecule inhibitors of reprogramming impediments improve naïve cell reprogramming beyond current methods. Collectively, this work defines the principles controlling the establishment of human naïve pluripotency and also provides new insights into mechanisms that destabilize and reconfigure cell identity during cell state transitions.


Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes , Complejo Represivo Polycomb 1 , Diferenciación Celular , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes/citología , Complejo Represivo Polycomb 1/metabolismo
6.
Stem Cells ; 28(1): 84-92, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19937754

RESUMEN

The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence-activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication.


Asunto(s)
Diferenciación Celular , Separación Celular/métodos , ARN Helicasas DEAD-box/genética , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Células Germinativas/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Regiones Promotoras Genéticas , Diferenciación Celular/genética , Línea Celular , Ensamble y Desensamble de Cromatina , Femenino , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Humanos , Meiosis , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transfección
7.
Stem Cells ; 28(4): 661-73, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20073085

RESUMEN

The generation of induced pluripotent stem cells (iPSC) has enormous potential for the development of patient-specific regenerative medicine. Human embryonic stem cells (hESC) are able to defend their genomic integrity by maintaining low levels of reactive oxygen species (ROS) through a combination of enhanced removal capacity and limited production of these molecules. Such limited ROS production stems partly from the small number of mitochondria present in hESC; thus, it was important to determine that human iPSC (hiPSC) generation is able to eliminate the extra mitochondria present in the parental fibroblasts (reminiscent of "bottleneck" situation after fertilization) and to show that hiPSC have antioxidant defenses similar to hESC. We were able to generate seven hiPSC lines from adult human dermal fibroblasts and have fully characterized two of those clones. Both hiPSC clones express pluripotency markers and are able to differentiate in vitro into cells belonging to all three germ layers. One of these clones is able to produce fully differentiated teratoma, whereas the other hiPSC clone is unable to silence the viral expression of OCT4 and c-MYC, produce fully differentiated teratoma, and unable to downregulate the expression of some of the pluripotency genes during the differentiation process. In spite of these differences, both clones show ROS stress defense mechanisms and mitochondrial biogenesis similar to hESC. Together our data suggest that, during the reprogramming process, certain cellular mechanisms are in place to ensure that hiPSC are provided with the same defense mechanisms against accumulation of ROS as the hESC.


Asunto(s)
Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Estrés Fisiológico , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Cariotipificación , Especies Reactivas de Oxígeno/metabolismo
8.
Stem Cell Reports ; 16(9): 2289-2304, 2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34450036

RESUMEN

Heterozygous mutations in HNF1B in humans result in a multisystem disorder, including pancreatic hypoplasia and diabetes mellitus. Here we used a well-controlled human induced pluripotent stem cell pancreatic differentiation model to elucidate the molecular mechanisms underlying HNF1B-associated diabetes. Our results show that lack of HNF1B blocks specification of pancreatic fate from the foregut progenitor (FP) stage, but HNF1B haploinsufficiency allows differentiation of multipotent pancreatic progenitor cells (MPCs) and insulin-secreting ß-like cells. We show that HNF1B haploinsufficiency impairs cell proliferation in FPs and MPCs. This could be attributed to impaired induction of key pancreatic developmental genes, including SOX11, ROBO2, and additional TEAD1 target genes whose function is associated with MPC self-renewal. In this work we uncover an exhaustive list of potential HNF1B gene targets during human pancreas organogenesis whose downregulation might underlie HNF1B-associated diabetes onset in humans, thus providing an important resource to understand the pathogenesis of this disease.


Asunto(s)
Diferenciación Celular/genética , Factor Nuclear 1-beta del Hepatocito/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Organogénesis/genética , Páncreas/embriología , Páncreas/metabolismo , Biomarcadores , Sistemas CRISPR-Cas , Linaje de la Célula/genética , Diabetes Mellitus/etiología , Susceptibilidad a Enfermedades , Técnica del Anticuerpo Fluorescente , Edición Génica , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Haploinsuficiencia , Factor Nuclear 1-beta del Hepatocito/metabolismo , Humanos , Inmunofenotipificación , Células Secretoras de Insulina/metabolismo , Transducción de Señal
9.
Stem Cells ; 26(12): 3075-85, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18802037

RESUMEN

Of all the cell types that can be obtained from the differentiation of embryonic stem cells, primordial germ cells are arguably the most fascinating, as they represent the in vitro completion of the reproductive cycle of the organism from which the embryonic stem cell line was derived. It is also possible to obtain these cells from embryos at an appropriate stage of development, but this process yields only small numbers that are not applicable to investigations of their epigenetic architecture. A considerable body of data has been generated from the differentiation of mouse embryonic stem cells to this cell type, but despite the demonstration of their presence in human embryoid bodies, there has been little progress toward methods of producing human primordial germ cells in useful numbers. We present here a robust protocol to differentiate two human embryonic stem cell lines (H9 and hES-NCL1) that maximizes the numbers of primordial germ cells that may be obtained using a simple fluorescence-activated cell sorting strategy for their isolation. These primordial germ cells demonstrate high-level expression of the germ cell-specific VASA gene and show removal of parental imprints and chromatin modification changes that support their primordial germ cell identity.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Células Germinativas/citología , Animales , Diferenciación Celular , Línea Celular , Cromatina/metabolismo , Islas de CpG , Epigénesis Genética , Citometría de Flujo , Células Germinativas/metabolismo , Humanos , Ratones , Microscopía Confocal/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección
10.
Cell Rep ; 24(2): 489-502, 2018 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-29996108

RESUMEN

The genetic basis of naive pluripotency maintenance and loss is a central question in embryonic stem cell biology. Here, we deploy CRISPR-knockout-based screens in mouse embryonic stem cells to interrogate this question through a genome-wide, non-biased approach using the Rex1GFP reporter as a phenotypic readout. This highly sensitive and efficient method identified genes in diverse biological processes and pathways. We uncovered a key role for negative regulators of mTORC1 in maintenance and exit from naive pluripotency and provided an integrated account of how mTORC1 activity influences naive pluripotency through Gsk3. Our study therefore reinforces Gsk3 as the central node and provides a comprehensive, data-rich resource that will improve our understanding of mechanisms regulating pluripotency and stimulate avenues for further mechanistic studies.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Técnicas de Inactivación de Genes , Genoma , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular/genética , Autorrenovación de las Células , Regulación del Desarrollo de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Células Madre Embrionarias de Ratones , Fenotipo , Transcriptoma/genética
11.
Cell Death Dis ; 9(2): 128, 2018 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-29374141

RESUMEN

Aplastic Anemia (AA) is a bone marrow failure (BMF) disorder, resulting in bone marrow hypocellularity and peripheral pancytopenia. Severe aplastic anemia (SAA) is a subset of AA defined by a more severe phenotype. Although the immunological nature of SAA pathogenesis is widely accepted, there is an increasing recognition of the role of dysfunctional hematopoietic stem cells in the disease phenotype. While pediatric SAA can be attributable to genetic causes, evidence is evolving on previously unrecognized genetic etiologies in a proportion of adults with SAA. Thus, there is an urgent need to better understand the pathophysiology of SAA, which will help to inform the course of disease progression and treatment options. We have derived induced pluripotent stem cell (iPSC) from three unaffected controls and three SAA patients and have shown that this in vitro model mimics two key features of the disease: (1) the failure to maintain telomere length during the reprogramming process and hematopoietic differentiation resulting in SAA-iPSC and iPSC-derived-hematopoietic progenitors with shorter telomeres than controls; (2) the impaired ability of SAA-iPSC-derived hematopoietic progenitors to give rise to erythroid and myeloid cells. While apoptosis and DNA damage response to replicative stress is similar between the control and SAA-iPSC-derived-hematopoietic progenitors, the latter show impaired proliferation which was not restored by eltrombopag, a drug which has been shown to restore hematopoiesis in SAA patients. Together, our data highlight the utility of patient specific iPSC in providing a disease model for SAA and predicting patient responses to various treatment modalities.


Asunto(s)
Anemia Aplásica/patología , Diferenciación Celular , Células Madre Hematopoyéticas/patología , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Acortamiento del Telómero , Benzoatos/farmacología , Estudios de Casos y Controles , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Células Madre Hematopoyéticas/metabolismo , Humanos , Hidrazinas/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Pirazoles/farmacología , Telomerasa/metabolismo , Telómero/metabolismo , Acortamiento del Telómero/efectos de los fármacos
12.
J Cell Biol ; 215(2): 187-202, 2016 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-27810911

RESUMEN

Human induced pluripotent stem cell (hiPSC) utility is limited by variations in the ability of these cells to undergo lineage-specific differentiation. We have undertaken a transcriptional comparison of human embryonic stem cell (hESC) lines and hiPSC lines and have shown that hiPSCs are inferior in their ability to undergo neuroectodermal differentiation. Among the differentially expressed candidates between hESCs and hiPSCs, we identified a mitochondrial protein, CHCHD2, whose expression seems to correlate with neuroectodermal differentiation potential of pluripotent stem cells. We provide evidence that hiPSC variability with respect to CHCHD2 expression and differentiation potential is caused by clonal variation during the reprogramming process and that CHCHD2 primes neuroectodermal differentiation of hESCs and hiPSCs by binding and sequestering SMAD4 to the mitochondria, resulting in suppression of the activity of the TGFß signaling pathway. Using CHCHD2 as a marker for assessing and comparing the hiPSC clonal and/or line differentiation potential provides a tool for large scale differentiation and hiPSC banking studies.


Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Madre Pluripotentes Inducidas/citología , Proteínas Mitocondriales/metabolismo , Placa Neural/citología , Factores de Transcripción/metabolismo , Apoptosis/genética , Secuencia de Bases , Línea Celular , Movimiento Celular/genética , Supervivencia Celular/genética , Reprogramación Celular/genética , Proteínas de Unión al ADN , Perfilación de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/metabolismo
13.
Stem Cells Transl Med ; 3(4): 416-23, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24591732

RESUMEN

Hypoplastic left heart syndrome (HLHS) is a serious congenital cardiovascular malformation resulting in hypoplasia or atresia of the left ventricle, ascending aorta, and aortic and mitral valves. Diminished flow through the left side of the heart is clearly a key contributor to the condition, but any myocardial susceptibility component is as yet undefined. Using recent advances in the field of induced pluripotent stem cells (iPSCs), we have been able to generate an iPSC model of HLHS malformation and characterize the properties of cardiac myocytes (CMs) differentiated from these and control-iPSC lines. Differentiation of HLHS-iPSCs to cardiac lineages revealed changes in the expression of key cardiac markers and a lower ability to give rise to beating clusters when compared with control-iPSCs and human embryonic stem cells (hESCs). HLHS-iPSC-derived CMs show a lower level of myofibrillar organization, persistence of a fetal gene expression pattern, and changes in commitment to ventricular versus atrial lineages, and they display different calcium transient patterns and electrophysiological responses to caffeine and ß-adrenergic antagonists when compared with hESC- and control-iPSC-derived CMs, suggesting that alternative mechanisms to release calcium from intracellular stores such as the inositol trisphosphate receptor may exist in HLHS in addition to the ryanodine receptor thought to function in control-iPSC-derived CMs. Together our findings demonstrate that CMs derived from an HLHS patient demonstrate a number of marker expression and functional differences to hESC/control iPSC-derived CMs, thus providing some evidence that cardiomyocyte-specific factors may influence the risk of HLHS.


Asunto(s)
Regulación de la Expresión Génica , Síndrome del Corazón Izquierdo Hipoplásico/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Proteínas Musculares/biosíntesis , Miocitos Cardíacos/metabolismo , Células Cultivadas , Humanos , Síndrome del Corazón Izquierdo Hipoplásico/patología , Células Madre Pluripotentes Inducidas/patología , Recién Nacido , Masculino , Miocitos Cardíacos/patología
14.
J Cell Sci ; 122(Pt 3): 401-13, 2009 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-19126678

RESUMEN

It is well documented that adipogenic differentiation of the cell is associated with downregulation of Wnt/beta-catenin signalling. Using preadipocytes and dermal fibroblasts, we have found that activation of the adipogenic program was associated with marked changes in the expression of nuclear beta-catenin-interacting partners, emerin and lamins A/C, to influence expression and activation of peroxisome proliferators-activated receptors gamma (PPARgamma). In addition, silencing of protein expression with siRNA revealed that beta-catenin and emerin influenced each other's levels of expression and the onset of adipogenesis, suggesting that changes in the expression of nuclear lamina proteins were intimately linked to the stability of beta-catenin. By contrast, dermal fibroblasts, which are emerin null, demonstrated increased nuclear accumulation of stable beta-catenin and constant lamin expression. This was also associated with an unusual adipogenic capacity of the cells, with adipogenesis occurring in the presence of activated beta-catenin but declining upon silencing of the protein expression with siRNA. We propose that the process of adipogenesis is affected by a dynamic link between complexes of emerin and lamins A/C at the nuclear envelope and nucleocytoplasmic distribution of beta-catenin, to influence cellular plasticity and differentiation.


Asunto(s)
Adipocitos/metabolismo , Adipogénesis/fisiología , Lamina Tipo A/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , beta Catenina/metabolismo , Adipocitos/citología , Animales , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/genética , Ratones , Proteínas Nucleares/genética , PPAR gamma/metabolismo , ARN Interferente Pequeño/metabolismo , beta Catenina/genética
15.
EMBO J ; 25(14): 3275-85, 2006 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-16858403

RESUMEN

Emerin is a type II inner nuclear membrane (INM) protein of unknown function. Emerin function is likely to be important because, when it is mutated, emerin promotes both skeletal muscle and heart defects. Here we show that one function of Emerin is to regulate the flux of beta-catenin, an important transcription coactivator, into the nucleus. Emerin interacts with beta-catenin through a conserved adenomatous polyposis coli (APC)-like domain. When GFP-emerin is expressed in HEK293 cells, beta-catenin is restricted to the cytoplasm and beta-catenin activity is inhibited. In contrast, expression of an emerin mutant, lacking its APC-like domain (GFP-emerinDelta), dominantly stimulates beta-catenin activity and increases nuclear accumulation of beta-catenin. Human fibroblasts that are null for emerin have an autostimulatory growth phenotype. This unusual growth phenotype arises through enhanced nuclear accumulation and activity of beta-catenin and can be replicated in wild-type fibroblasts by transfection with constitutively active beta-catenin. Our results support recent findings that suggest that INM proteins can influence signalling pathways by restricting access of transcription coactivators to the nucleus.


Asunto(s)
Proteínas de la Membrana/fisiología , Membrana Nuclear/fisiología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Timopoyetinas/fisiología , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismo , Línea Celular , Células Cultivadas , Humanos , Distrofia Muscular de Emery-Dreifuss/metabolismo , Transducción de Señal/fisiología , Transactivadores/fisiología
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