RESUMEN
Eukaryotic initiation factor 5A (eIF5A), is an essential protein that requires a unique amino acid, hypusine, for its activity. Hypusine is formed exclusively in eIF5A post-translationally via two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase. Each of the genes encoding these proteins, Eif5a, Dhps, and Dohh, is required for mouse embryonic development. Variants in EIF5A or DHPS were recently identified as the genetic basis underlying certain rare neurodevelopmental disorders in humans. To investigate the roles of eIF5A and DHPS in brain development, we generated four conditional KO mouse strains using the Emx1-Cre or Camk2a-Cre strains and examined the effects of temporal- and region-specific deletion of Eif5a or Dhps. The conditional deletion of Dhps or Eif5a by Emx1 promotor-driven Cre expression (E9.5, in the cortex and hippocampus) led to gross defects in forebrain development, reduced growth, and premature death. On the other hand, the conditional deletion of Dhps or Eif5a by Camk2a promoter-driven Cre expression (postnatal, mainly in the CA1 region of the hippocampus) did not lead to global developmental defects; rather, these KO animals exhibited severe impairment in spatial learning, contextual learning, and memory when subjected to the Morris water maze and a contextual learning test. In both models, the Dhps-KO mice displayed more severe impairment than their Eif5a-KO counterparts. The observed defects in the brain, global development, or cognitive functions most likely result from translation errors due to a deficiency in active, hypusinated eIF5A. Our study underscores the important roles of eIF5A and DHPS in neurodevelopment.
Asunto(s)
Corteza Cerebelosa/metabolismo , Cognición , Hipocampo/metabolismo , Oxigenasas de Función Mixta/metabolismo , Neurogénesis , Neuronas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Ratones , Ratones Noqueados , Oxigenasas de Función Mixta/genética , Especificidad de Órganos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Hypusine is formed post-translationally from lysine and is found in a single cellular protein, eukaryotic translation initiation factor-5A (eIF5A), and its homolog eIF5A2. Biosynthesis of hypusine is a two-step reaction involving the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). eIF5A is highly conserved throughout eukaryotic evolution and plays a role in mRNA translation, cellular proliferation, cellular differentiation, and inflammation. DHPS is also highly conserved and is essential for life, as Dhps-null mice are embryonic lethal. Using exome sequencing, we identified rare biallelic, recurrent, predicted likely pathogenic variants in DHPS segregating with disease in five affected individuals from four unrelated families. These individuals have similar neurodevelopmental features that include global developmental delay and seizures. Two of four affected females have short stature. All five affected individuals share a recurrent missense variant (c.518A>G [p.Asn173Ser]) in trans with a likely gene disrupting variant (c.1014+1G>A, c.912_917delTTACAT [p.Tyr305_Ile306del], or c.1A>G [p.Met1?]). cDNA studies demonstrated that the c.1014+1G>A variant causes aberrant splicing. Recombinant DHPS enzyme harboring either the p.Asn173Ser or p.Tyr305_Ile306del variant showed reduced (20%) or absent in vitro activity, respectively. We co-transfected constructs overexpressing HA-tagged DHPS (wild-type or mutant) and GFP-tagged eIF5A into HEK293T cells to determine the effect of these variants on hypusine biosynthesis and observed that the p.Tyr305_Ile306del and p.Asn173Ser variants resulted in reduced hypusination of eIF5A compared to wild-type DHPS enzyme. Our data suggest that rare biallelic variants in DHPS result in reduced enzyme activity that limits the hypusination of eIF5A and are associated with a neurodevelopmental disorder.
Asunto(s)
Genes Recesivos/genética , Lisina/análogos & derivados , Mutación , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Alelos , Secuencia de Aminoácidos , Niño , Preescolar , Discapacidades del Desarrollo/enzimología , Discapacidades del Desarrollo/genética , Femenino , Haplotipos , Humanos , Lisina/biosíntesis , Masculino , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Linaje , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Convulsiones/enzimología , Convulsiones/genética , Adulto Joven , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Pancreatic diseases including diabetes and exocrine insufficiency would benefit from therapies that reverse cellular loss and/or restore cellular mass. The identification of molecular pathways that influence cellular growth is therefore critical for future therapeutic generation. Deoxyhypusine synthase (DHPS) is an enzyme that post-translationally modifies and activates the mRNA translation factor eukaryotic initiation factor 5A (eIF5A). Previous work demonstrated that the inhibition of DHPS impairs zebrafish exocrine pancreas development; however, the link between DHPS, eIF5A, and regulation of pancreatic organogenesis remains unknown. Herein we identified that the conditional deletion of either Dhps or Eif5a in the murine pancreas results in the absence of acinar cells. Because DHPS catalyzes the activation of eIF5A, we evaluated and uncovered a defect in mRNA translation concomitant with defective production of proteins that influence cellular development. Our studies reveal a heretofore unappreciated role for DHPS and eIF5A in the synthesis of proteins required for cellular development and function.
Asunto(s)
Lisina/análogos & derivados , Organogénesis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/fisiología , Páncreas Exocrino/citología , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismo , Animales , Proliferación Celular , Femenino , Lisina/biosíntesis , Masculino , Ratones , Ratones Noqueados , Páncreas Exocrino/metabolismo , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Intestinal hormone-producing cells represent the largest endocrine system in the body, but remarkably little is known about enteroendocrine cell type specification in the embryo and adult. We analyzed stage- and cell type-specific deletions of Nkx2.2 and its functional domains in order to characterize its role in the development and maintenance of enteroendocrine cell lineages in the mouse duodenum and colon. Although Nkx2.2 regulates enteroendocrine cell specification in the duodenum at all stages examined, it controls the differentiation of progressively fewer enteroendocrine cell populations when deleted from Ngn3(+) progenitor cells or in the adult duodenum. During embryonic development Nkx2.2 regulates all enteroendocrine cell types, except gastrin and preproglucagon. In developing Ngn3(+) enteroendocrine progenitor cells, Nkx2.2 is not required for the specification of neuropeptide Y and vasoactive intestinal polypeptide, indicating that a subset of these cell populations derive from an Nkx2.2-independent lineage. In adult duodenum, Nkx2.2 becomes dispensable for cholecystokinin and secretin production. In all stages and Nkx2.2 mutant conditions, serotonin-producing enterochromaffin cells were the most severely reduced enteroendocrine lineage in the duodenum and colon. We determined that the transcription factor Lmx1a is expressed in enterochromaffin cells and functions downstream of Nkx2.2. Lmx1a-deficient mice have reduced expression of Tph1, the rate-limiting enzyme for serotonin biosynthesis. These data clarify the function of Nkx2.2 in the specification and homeostatic maintenance of enteroendocrine populations, and identify Lmx1a as a novel enterochromaffin cell marker that is also essential for the production of the serotonin biosynthetic enzyme Tph1.
Asunto(s)
Linaje de la Célula , Células Enterocromafines/citología , Células Enteroendocrinas/citología , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Serotonina/biosíntesis , Factores de Transcripción/metabolismo , Envejecimiento/metabolismo , Animales , Biomarcadores/metabolismo , Linaje de la Célula/genética , Colon/metabolismo , Duodeno/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/química , Ratones Endogámicos C57BL , Modelos Biológicos , Mutación/genética , Reacción en Cadena de la Polimerasa , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Células Madre/citología , Factores de Transcripción/química , Proteínas de Pez CebraRESUMEN
AIMS: Our current understanding of the pathogenesis of type 1 diabetes (T1D) arose, in large part, from studies using the non-obese diabetic (NOD) mouse model. In the present study, we chose a human-focused method to investigate T1D disease mechanisms and potential targets for therapeutic intervention by directly analysing human donor pancreatic islets from individuals with T1D. MATERIALS AND METHODS: We obtained islets from a young individual with T1D for 3 years and from an older individual with T1D for 27 years and performed unbiased functional genomic analysis by high-depth RNA sequencing; the T1D islets were compared with islets isolated from 3 non-diabetic donors. RESULTS: The islets procured from these T1D donors represent a unique opportunity to identify gene expression changes in islets after significantly different disease duration. Data analysis identified several inflammatory pathways up-regulated in short-duration disease, which notably included many components of innate immunity. As proof of concept for translation, one of the pathways, governed by IL-23(p19), was selected for further study in NOD mice because of ongoing human trials of biologics against this target for different indications. A mouse monoclonal antibody directed against IL-23(p19) when administered to NOD mice resulted in a significant reduction in incidence of diabetes. CONCLUSION: While the sample size for this study is small, our data demonstrate that the direct analysis of human islets provides a greater understanding of human disease. These data, together with the analysis of an expanded cohort to be obtained by future collaborative efforts, might result in the identification of promising novel targets for translation into effective therapeutic interventions for human T1D, with the added benefit of repurposing known biologicals for use in different indications.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Regulación de la Expresión Génica , Islotes Pancreáticos/metabolismo , Adulto , Animales , Anticuerpos Monoclonales/uso terapéutico , Cadáver , Niño , Análisis por Conglomerados , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 1/prevención & control , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata , Subunidad p19 de la Interleucina-23/antagonistas & inhibidores , Subunidad p19 de la Interleucina-23/genética , Subunidad p19 de la Interleucina-23/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/patología , Masculino , Ratones Endogámicos NOD , Prueba de Estudio Conceptual , Donantes de TejidosRESUMEN
As one of the key nutrient sensors, insulin signaling plays an important role in integrating environmental energy cues with organism growth. In adult organisms, relative insufficiency of insulin signaling induces compensatory expansion of insulin-secreting pancreatic beta (ß) cells. However, little is known about how insulin signaling feedback might influence neogenesis of ß cells during embryonic development. Using genetic approaches and a unique cell transplantation system in developing zebrafish, we have uncovered a novel role for insulin signaling in the negative regulation of pancreatic progenitor cell differentiation. Blocking insulin signaling in the pancreatic progenitors hastened the expression of the essential ß cell genes insulin and pdx1, and promoted ß cell fate at the expense of alpha cell fate. In addition, loss of insulin signaling promoted ß cell regeneration and destabilization of alpha cell character. These data indicate that insulin signaling constitutes a tunable mechanism for ß cell compensatory plasticity during early development. Moreover, using a novel blastomere-to-larva transplantation strategy, we found that loss of insulin signaling in endoderm-committed blastomeres drove their differentiation into ß cells. Furthermore, the extent of this differentiation was dependent on the function of the ß cell mass in the host. Altogether, our results indicate that modulation of insulin signaling will be crucial for the development of ß cell restoration therapies for diabetics; further clarification of the mechanisms of insulin signaling in ß cell progenitors will reveal therapeutic targets for both in vivo and in vitro ß cell generation.
Asunto(s)
Diferenciación Celular , Retroalimentación Fisiológica , Insulina/metabolismo , Islotes Pancreáticos/embriología , Regeneración , Transducción de Señal , Células Madre/citología , Animales , Blastómeros/citología , Blastómeros/metabolismo , Blastómeros/trasplante , Linaje de la Célula , Endodermo/citología , Endodermo/embriología , Endodermo/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/citología , Islotes Pancreáticos/citología , Receptor de Insulina/metabolismo , Transactivadores/metabolismo , Pez Cebra/embriologíaRESUMEN
During pancreatic development, transcription factor cascades gradually commit precursor populations to the different endocrine cell fate pathways. Although mutational analyses have defined the functions of many individual pancreatic transcription factors, the integrative transcription factor networks required to regulate lineage specification, as well as their sites of action, are poorly understood. In this study, we investigated where and how the transcription factors Nkx2.2 and Neurod1 genetically interact to differentially regulate endocrine cell specification. In an Nkx2.2 null background, we conditionally deleted Neurod1 in the Pdx1+ pancreatic progenitor cells, the Neurog3+ endocrine progenitor cells, or the glucagon+ alpha cells. These studies determined that, in the absence of Nkx2.2 activity, removal of Neurod1 from the Pdx1+ or Neurog3+ progenitor populations is sufficient to reestablish the specification of the PP and epsilon cell lineages. Alternatively, in the absence of Nkx2.2, removal of Neurod1 from the Pdx1+ pancreatic progenitor population, but not the Neurog3+ endocrine progenitor cells, restores alpha cell specification. Subsequent in vitro reporter assays demonstrated that Nkx2.2 represses Neurod1 in alpha cells. Based on these findings, we conclude that, although Nkx2.2 and Neurod1 are both necessary to promote beta cell differentiation, Nkx2.2 must repress Neurod1 in a Pdx1+ pancreatic progenitor population to appropriately commit a subset of Neurog3+ endocrine progenitor cells to the alpha cell lineage. These results are consistent with the proposed idea that Neurog3+ endocrine progenitor cells represent a heterogeneous population of unipotent cells, each restricted to a particular endocrine lineage.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Proteínas de Homeodominio , Páncreas , Factores de Transcripción , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Células Secretoras de Glucagón/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ratones , Proteínas Nucleares , Páncreas/citología , Páncreas/crecimiento & desarrollo , Páncreas/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Pez CebraRESUMEN
BACKGROUND: All vertebrate peripheral nerves connect the central nervous system (CNS) with targets in the periphery and are composed of axons, layers of ensheathing glia and connective tissue. Although the structure of these conduits is well established, very little is known about the origin and developmental roles of some of their elements. One understudied component, the perineurium, ensheaths nerve fascicles and is a component of the blood-nerve-barrier. In zebrafish, the motor nerve perineurium is composed of CNS-derived nkx2.2a(+) perineurial glia, which establish the motor exit point (MEP) during development. To determine if mouse perineurial cells also originate within the CNS and perform a similar function, we created a Nkx2.2:EGFP transgenic reporter line. RESULTS: In conjunction with RNA expression analysis and antibody labeling, we observed Nkx2.2(+) cells along peripheral motor nerves at all stages of development and in adult tissue. Additionally, in mice lacking Nkx2.2, we demonstrate that Nkx2.2(+) perineurial glia are essential for motor nerve development and Schwann cell differentiation. CONCLUSIONS: Our studies reveal that a subset of mouse perineurial cells are CNS-derived, express Nkx2.2, and are essential for motor nerve development. This work highlights an under-appreciated but essential contribution of CNS-derived cells to the development of the mammalian peripheral nervous system (PNS).
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Homeodominio/metabolismo , Neuronas Motoras/citología , Neuroglía/citología , Nervios Periféricos/citología , Células de Schwann/citología , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Axones/fisiología , Línea Celular , Proteína Homeobox Nkx-2.2 , Ratones , Neuronas Motoras/metabolismo , Neuroglía/metabolismo , Nervios Periféricos/metabolismo , Células de Schwann/metabolismo , Proteínas de Pez CebraRESUMEN
The exocrine and endocrine are functionally distinct compartments of the pancreas that have traditionally been studied as separate entities. However, studies of embryonic development, adult physiology, and disease pathogenesis suggest there may be critical communication between exocrine and endocrine cells. In fact, the incidence of the endocrine disease diabetes secondary to exocrine disease/dysfunction ranges from 25% to 80%, depending on the type and severity of the exocrine pathology. Therefore, it is necessary to investigate how exocrine-endocrine "crosstalk" may impact pancreatic function. In this article, we discuss common exocrine diseases, including cystic fibrosis, acute, hereditary, and chronic pancreatitis, and the impact of these exocrine diseases on endocrine function. Additionally, we review how obesity and fatty pancreas influence exocrine function and the impact on cellular communication between the exocrine and endocrine compartments. Interestingly, in all pathologies, there is evidence that signals from the exocrine disease contribute to endocrine dysfunction and the progression to diabetes. Continued research efforts to identify the mechanisms that underlie the crosstalk between various cell types in the pancreas are critical to understanding normal pancreatic physiology as well as disease states. © 2024 American Physiological Society. Compr Physiol 14:5371-5387, 2024.
Asunto(s)
Páncreas Exocrino , Enfermedades Pancreáticas , Humanos , Animales , Enfermedades Pancreáticas/fisiopatología , Enfermedades Pancreáticas/patología , Enfermedades Pancreáticas/metabolismo , Páncreas Exocrino/fisiopatología , Páncreas Exocrino/metabolismo , Páncreas Exocrino/patología , Páncreas/fisiopatología , Páncreas/patología , Sistema Endocrino/fisiopatología , Sistema Endocrino/fisiologíaRESUMEN
As professional secretory cells, ß-cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic ß-cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional ß-cells is not well defined. In this study, we have identified a translational regulatory mechanism mediated by the specialized mRNA translation factor eukaryotic initiation factor 5A (eIF5A), which facilitates the maintenance of ß-cell identity and function. The mRNA translation function of eIF5A is only active when it is posttranslationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of ß-cell DHPS in mice reduces the synthesis of proteins critical to ß-cell identity and function at the stage of ß-cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the ß-cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand.
Asunto(s)
Células Secretoras de Insulina , Factores de Iniciación de Péptidos , Animales , Ratones , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Células Secretoras de Insulina/metabolismo , Biosíntesis de Proteínas , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismoRESUMEN
Nkx2.2 is a homeobox transcription factor that is expressed in the pancreas, intestine and central nervous system (CNS) during embryogenesis and in the adult. In mice, global deletion of Nkx2.2 results in cell mis-specification in each of the tissues where it is expressed, and the null mice die as neonates with severe hyperglycemia. Although a whole body knockout demonstrates the importance of Nkx2.2 in cell specification and postnatal viability, it precludes assessment of the cell-autonomous and postnatal functions of Nkx2.2. In this study we report the generation and functional characterization of mice encoding a conditional allele of Nkx2.2. We demonstrate the functional integrity of the conditional Nkx2.2 allele and report successful in vivo deletion using a pancreas-specific Cre recombinase. The pancreas-specific deletion of Nkx2.2 results in similar defects found in the Nkx2.2 null pancreas and the mice die shortly after birth, demonstrating that the neonatal lethality associated with the null allele is caused by pancreatic islet dysfunction. The generation of a conditional Nkx2.2 allele provides an important tool for identifying the cell-autonomous and postnatal activities of Nkx2.2 in establishing and maintaining cell type identities and functions in the pancreas, intestine and CNS.
Asunto(s)
Proteínas de Homeodominio/genética , Ratones Transgénicos/genética , Páncreas/metabolismo , Factores de Transcripción/genética , Animales , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Proteína Homeobox Nkx-2.2 , Hibridación in Situ , Integrasas , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas de Pez CebraRESUMEN
DHPS deficiency is a rare genetic disease caused by biallelic hypomorphic variants in the Deoxyhypusine synthase (DHPS) gene. The DHPS enzyme functions in mRNA translation by catalyzing the post-translational modification, and therefore activation, of eukaryotic initiation factor 5A (eIF5A). The observed clinical outcomes associated with human mutations in DHPS include developmental delay, intellectual disability, and seizures. Therefore, to increase our understanding of this rare disease, it is critical to determine the mechanisms by which mutations in DHPS alter neurodevelopment. In this study, we have generated patient-derived lymphoblast cell lines and demonstrated that human DHPS variants alter DHPS protein abundance and impair enzyme function. Moreover, we observe a shift in the abundance of the post-translationally modified forms of eIF5A; specifically, an increase in the nuclear localized acetylated form (eIF5AAcK47) and concomitant decrease in the cytoplasmic localized hypusinated form (eIF5AHYP). Generation and characterization of a mouse model with a genetic deletion of Dhps in the brain at birth shows that loss of hypusine biosynthesis impacts neuronal function due to impaired eIF5AHYP-dependent mRNA translation; this translation defect results in altered expression of proteins required for proper neuronal development and function. This study reveals new insight into the biological consequences and molecular impact of human DHPS deficiency and provides valuable information toward the goal of developing treatment strategies for this rare disease.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , Factores de Iniciación de Péptidos , Enfermedades Raras , Animales , Humanos , Recién Nacido , Ratones , Homeostasis/genética , Mutación , Factores de Iniciación de Péptidos/genética , Procesamiento Proteico-Postraduccional/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
As professional secretory cells, beta cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic beta cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional beta cells is not well defined. In this study, we have identified a translational regulatory mechanism in the beta cell driven by the specialized mRNA translation factor, eukaryotic initiation factor 5A (eIF5A), which facilitates beta cell maturation. The mRNA translation function of eIF5A is only active when it is post-translationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of beta cell DHPS in mice reduces the synthesis of proteins critical to beta cell identity and function at the stage of beta cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the beta cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand. ARTICLE HIGHLIGHTS: Pancreatic beta cells are professional secretory cells that require adaptable mRNA translation for the rapid, inducible synthesis of proteins, including insulin, in response to changing metabolic cues. Our previous work in the exocrine pancreas showed that development and function of the acinar cells, which are also professional secretory cells, is regulated at the level of mRNA translation by a specialized mRNA translation factor, eIF5A HYP . We hypothesized that this translational regulation, which can be a response to stress such as changes in growth or metabolism, may also occur in beta cells. Given that the mRNA translation function of eIF5A is only active when the factor is post-translationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS), we asked the question: does DHPS/eIF5A HYP regulate the formation and maintenance of functional beta cells? We discovered that in the absence of beta cell DHPS in mice, eIF5A is not hypusinated (activated), which leads to a reduction in the synthesis of critical beta cell proteins that interrupts pathways critical for identity and function. This translational regulation occurs at weaning age, which is a stage of cellular stress and maturation for the beta cell. Therefore without DHPS/eIF5A HYP , beta cells do not mature and mice progress to hyperglycemia and diabetes. Our findings suggest that secretory cells have a mechanism to regulate mRNA translation during times of cellular stress. Our work also implies that driving an increase in mRNA translation in the beta cell might overcome or possibly reverse the beta cell defects that contribute to early dysfunction and the progression to diabetes.
RESUMEN
In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing ß cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with ß cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during ß cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125-1,000 mg/m2) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve ß cell function in T1D through islet cell-autonomous effects.
Asunto(s)
Diabetes Mellitus Tipo 1 , Humanos , Ratones , Animales , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Inhibidores de la Ornitina Descarboxilasa/farmacología , Eflornitina/farmacología , Eflornitina/uso terapéutico , Putrescina/metabolismoRESUMEN
The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.
Asunto(s)
Diabetes Mellitus , Islotes Pancreáticos , Páncreas Exocrino , Enfermedades Pancreáticas , Humanos , Diabetes Mellitus/metabolismo , Páncreas , Enfermedades Pancreáticas/metabolismoRESUMEN
Nkx2.2 and Arx are essential pancreatic transcription factors. Nkx2.2 is necessary for the appropriate specification of the islet alpha, beta, PP and epsilon cell lineages, whereas Arx is required to form the correct ratio of alpha, beta, delta and PP cells. To begin to understand the cooperative functions of Nkx2.2 and Arx in the development of endocrine cell lineages, we generated progenitor cell-specific deletions of Arx on the Nkx2.2 null background. The analysis of these mutants demonstrates that expansion of the ghrelin cell population in the Nkx2.2 null pancreas is not dependent on Arx; however, Arx is necessary for the upregulation of ghrelin mRNA levels in Nkx2.2 mutant epsilon cells. Alternatively, in the absence of Arx, delta cell numbers are increased and Nkx2.2 becomes essential for the repression of somatostatin gene expression. Interestingly, the dysregulation of ghrelin and somatostatin expression in the Nkx2.2/Arx compound mutant (Nkx2.2(null);Arx(Δpanc)) results in the appearance of ghrelin+/somatostatin+ co-expressing cells. These compound mutants also revealed a genetic interaction between Nkx2.2 and Arx in the regulation of the PP cell lineage; the PP cell population is reduced when Nkx2.2 is deleted but is restored back to wildtype numbers in the Nkx2.2(null);Arx(Δpanc) mutant. Moreover, conditional deletion of Arx in specific pancreatic cell populations established that the functions of Arx are necessary in the Neurog3+ endocrine progenitors. Together, these experiments identify novel genetic interactions between Nkx2.2 and Arx within the endocrine progenitor cells that ensure the correct specification and regulation of endocrine hormone-producing cells.
Asunto(s)
Proteínas de Homeodominio/genética , Páncreas/citología , Hormonas Pancreáticas/metabolismo , Factores de Transcripción/genética , Animales , Linaje de la Célula , Proteína Homeobox Nkx-2.2 , Ratones , Páncreas/metabolismo , Proteínas de Pez CebraRESUMEN
Diabetes mellitus, a disease that affects nearly 536.6 million people worldwide, is characterized by the death or dysfunction of insulin-producing beta cells of the pancreas. The beta cells are found within the islets of Langerhans, which are composed of multiple hormone-producing endocrine cells including the alpha (glucagon), delta (somatostatin), PP (pancreatic polypeptide), and epsilon (ghrelin) cells. There is direct evidence that physical and paracrine interactions between the cells in the islet facilitate and support beta cell function. However, communication between endocrine and exocrine cells in the pancreas may also directly impact beta cell growth and function. Herein we review literature that contributes to the view that "crosstalk" between neighboring cells within the pancreas influences beta cell growth and function and the maintenance of beta cell health.
Asunto(s)
Diabetes Mellitus , Células Endocrinas , Células Secretoras de Insulina , Glucagón , Humanos , PáncreasRESUMEN
ABSTRACT: The "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.
Asunto(s)
Diabetes Mellitus , Islotes Pancreáticos , Páncreas Exocrino , Enfermedades Pancreáticas , Humanos , Diabetes Mellitus/terapia , Diabetes Mellitus/metabolismo , Islotes Pancreáticos/metabolismo , Páncreas/metabolismo , Páncreas Exocrino/metabolismo , Enfermedades Pancreáticas/diagnóstico , Enfermedades Pancreáticas/terapia , Enfermedades Pancreáticas/metabolismoRESUMEN
BACKGROUND: The creation of a complete genome-wide map of transcription factor binding sites is essential for understanding gene regulatory networks in vivo. However, current prediction methods generally rely on statistical models that imperfectly model transcription factor binding. Generation of new prediction methods that are based on protein binding data, but do not rely on these models may improve prediction sensitivity and specificity. RESULTS: We propose a method for predicting transcription factor binding sites in the genome by directly mapping data generated from protein binding microarrays (PBM) to the genome and calculating a moving average of several overlapping octamers. Using this unique algorithm, we predicted binding sites for the essential pancreatic islet transcription factor Nkx2.2 in the mouse genome and confirmed >90% of the tested sites by EMSA and ChIP. Scores generated from this method more accurately predicted relative binding affinity than PWM based methods. We have also identified an alternative core sequence recognized by the Nkx2.2 homeodomain. Furthermore, we have shown that this method correctly identified binding sites in the promoters of two critical pancreatic islet ß-cell genes, NeuroD1 and insulin2, that were not predicted by traditional methods. Finally, we show evidence that the algorithm can also be applied to predict binding sites for the nuclear receptor Hnf4α. CONCLUSIONS: PBM-mapping is an accurate method for predicting Nkx2.2 binding sites and may be widely applicable for the creation of genome-wide maps of transcription factor binding sites.
Asunto(s)
Sitios de Unión , Biología Computacional/métodos , Proteínas de Homeodominio/genética , Análisis por Matrices de Proteínas , Factores de Transcripción/genética , Algoritmos , Animales , Genoma , Proteína Homeobox Nkx-2.2 , Ratones , Páncreas , Regiones Promotoras Genéticas , Unión Proteica , Análisis de Regresión , Proteínas de Pez CebraRESUMEN
During forebrain development, Mash1 directs gamma-aminobutyric acid (GABA)ergic neuron differentiation ventrally in the ganglionic eminences. Repression of Mash1 in the cortex is necessary to prevent the formation of GABAergic interneurons. Negative regulation of Mash1 has been attributed to members of the Neurogenin family; the genetic ablation of Neurogenin2 (Ngn2) leads to the derepression of Mash1 and the formation of ectopic GABAergic neurons in the cortex. We have developed an in vitro system to clarify the importance of NeuroD proteins in the Mash1 regulatory pathway. Using a neurosphere culture system, we show that the downstream effectors of the Ngn2 pathway NeuroD1 and NeuroD2 can abrogate GABAergic differentiation directed by Mash1. The ectopic expression of either of these genes in Mash1-expressing cells derived from the lateral ganglionic eminence, independently downregulate Mash1 expression without affecting expression of distal less homeodomain genes. This results in a complete loss of the GABAergic phenotype. Moreover, we demonstrate that ectopic expression of Mash1 in cortical progenitors is sufficient to phenocopy the loss of Ngn2 and strongly enhances ectopic GABAergic differentiation. Collectively, our results define the compensatory and cross-regulatory mechanisms that exist among basic helix-loop-helix transcription factors during neuronal fate specification.