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1.
Elife ; 102021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34009124

RESUMO

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.


Assuntos
Perfilação da Expressão Gênica , Heterogeneidade Genética , Ductos Pancreáticos/citologia , Análise de Célula Única , Transcriptoma , Animais , Linhagem Celular , Separação Celular , Dano ao DNA , Bases de Dados Genéticas , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Feminino , Geminina/genética , Geminina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfogênese , Osteopontina/genética , Osteopontina/metabolismo , Ductos Pancreáticos/metabolismo , Pancreatite Crônica/genética , Pancreatite Crônica/metabolismo , Pancreatite Crônica/patologia , Fenótipo , RNA-Seq
2.
Nat Commun ; 11(1): 2742, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488111

RESUMO

Next generation sequencing studies have highlighted discrepancies in ß-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human ß-cells postnatally, while its expression is restricted to embryonic and neo-natal ß-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to ß-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide-positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology.


Assuntos
Células Secretoras de Insulina/metabolismo , Fator de Transcrição MafB/genética , Fator de Transcrição MafB/metabolismo , Células Estreladas do Pâncreas/metabolismo , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Feminino , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Células-Tronco , Transcriptoma
3.
J Clin Invest ; 129(10): 4124-4137, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31265435

RESUMO

Pancreatic beta cells (ß-cells) differentiate during fetal life, but only postnatally acquire the capacity for glucose-stimulated insulin secretion (GSIS). How this happens is not clear. In exploring what molecular mechanisms drive the maturation of ß-cell function, we found that the control of cellular signaling in ß-cells fundamentally switched from the nutrient sensor target of rapamycin (mTORC1) to the energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK), and that this was critical for functional maturation. Moreover, AMPK was activated by the dietary transition taking place during weaning, and this in turn inhibited mTORC1 activity to drive the adult ß-cell phenotype. While forcing constitutive mTORC1 signaling in adult ß-cells relegated them to a functionally immature phenotype with characteristic transcriptional and metabolic profiles, engineering the switch from mTORC1 to AMPK signaling was sufficient to promote ß-cell mitochondrial biogenesis, a shift to oxidative metabolism, and functional maturation. We also found that type 2 diabetes, a condition marked by both mitochondrial degeneration and dysregulated GSIS, was associated with a remarkable reversion of the normal AMPK-dependent adult ß-cell signature to a more neonatal one characterized by mTORC1 activation. Manipulating the way in which cellular nutrient signaling pathways regulate ß-cell metabolism may thus offer new targets to improve ß-cell function in diabetes.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Secreção de Insulina/genética , Células Secretoras de Insulina/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Knockout
4.
Nat Cell Biol ; 21(6): 792, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30914825

RESUMO

In the version of this article originally published, the Gene Expression Omnibus (GEO) accession number listed in the data availability section was incorrectly given as GSE10979 instead of GSE109795. The sentence should read "RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE109795," and the code should link to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE109795. The error has been corrected in the HTML and PDF versions of the paper.

5.
Immunity ; 50(2): 362-377.e6, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30709738

RESUMO

Regulatory T (Treg) cells maintain immune tolerance through the master transcription factor forkhead box P3 (FOXP3), which is crucial for Treg cell function and homeostasis. We identified an IPEX (immune dysregulation polyendocrinopathy enteropathy X-linked) syndrome patient with a FOXP3 mutation in the domain swap interface of the protein. Recapitulation of this Foxp3 variant in mice led to the development of an autoimmune syndrome consistent with an unrestrained T helper type 2 (Th2) immune response. Genomic analysis of Treg cells by RNA-sequencing, Foxp3 chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-sequencing), and H3K27ac-HiChIP revealed a specific de-repression of the Th2 transcriptional program leading to the generation of Th2-like Treg cells that were unable to suppress extrinsic Th2 cells. Th2-like Treg cells showed increased intra-chromosomal interactions in the Th2 locus, leading to type 2 cytokine production. These findings identify a direct role for Foxp3 in suppressing Th2-like Treg cells and implicate additional pathways that could be targeted to restrain Th2 trans-differentiated Treg cells.


Assuntos
Fatores de Transcrição Forkhead/imunologia , Mutação , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Criança , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poliendocrinopatias Autoimunes/genética , Poliendocrinopatias Autoimunes/imunologia , Poliendocrinopatias Autoimunes/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th2/metabolismo
6.
Nat Cell Biol ; 21(2): 263-274, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30710150

RESUMO

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional ß cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and ß cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature ß-like cells to form islet-sized enriched ß-clusters (eBCs). eBCs display physiological properties analogous to primary human ß cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell clustering induces metabolic maturation by driving mitochondrial oxidative respiration, a process central to stimulus-secretion coupling in mature ß cells. eBCs display glucose-stimulated insulin secretion as early as three days after transplantation in mice. In summary, replicating aspects of endocrine cell clustering permits the generation of stem-cell-derived ß cells that resemble their endogenous counterparts.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células Endócrinas/citologia , Fibroblastos/citologia , Células-Tronco Embrionárias Humanas/citologia , Células Secretoras de Insulina/citologia , Animais , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Células Endócrinas/fisiologia , Fibroblastos/fisiologia , Glucose/farmacologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/citologia , Camundongos , Mitocôndrias/metabolismo
7.
Cell Rep ; 18(11): 2635-2650, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28297668

RESUMO

The nuclear cap-binding complex (CBC) stimulates processing reactions of capped RNAs, including their splicing, 3'-end formation, degradation, and transport. CBC effects are particular for individual RNA families, but how such selectivity is achieved remains elusive. Here, we analyze three main CBC partners known to impact different RNA species. ARS2 stimulates 3'-end formation/transcription termination of several transcript types, ZC3H18 stimulates degradation of a diverse set of RNAs, and PHAX functions in pre-small nuclear RNA/small nucleolar RNA (pre-snRNA/snoRNA) transport. Surprisingly, these proteins all bind capped RNAs without strong preferences for given transcripts, and their steady-state binding correlates poorly with their function. Despite this, PHAX and ZC3H18 compete for CBC binding and we demonstrate that this competitive binding is functionally relevant. We further show that CBC-containing complexes are short lived in vivo, and we therefore suggest that RNA fate involves the transient formation of mutually exclusive CBC complexes, which may only be consequential at particular checkpoints during RNA biogenesis.


Assuntos
Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , RNA/metabolismo , Células HEK293 , Células HeLa , Humanos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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